Compounds,compositions, and methods for the treatment of disease

ABSTRACT

Disclosed are compounds and compositions for the activation or induction of expression of a pattern recognition receptor (e.g., STING, RIG-I, MDA5), and methods of use thereof.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. provisionalpatent application Nos. 62/359,039, filed Jul. 6, 2016; 62/363,118,filed Jul. 15, 2016; 62/403,530, filed Oct. 3, 2016; 62/411,424, filedOct. 21, 2016; 62/444,141, filed Jan. 9, 2017; 62/462,679, filed Feb.23, 2017; 62/470,746, filed Mar. 13, 2017; and 62/508,846, May 19, 2017;the contents of each of which are hereby incorporated by reference intheir entireties.

FIELD OF DISCLOSURE

This disclosure relates to compounds and compositions that activate theinnate immune defense system and induce expression of patternrecognition receptors in a host, as well as methods of use for thetreatment of a proliferative disease (e.g., cancer).

BACKGROUND OF DISCLOSURE

A key feature of the innate immune system is the recognition andelimination of foreign substances. Identification of these pathogenicinvaders occurs through host recognition of evolutionarily conservedmicrobial structures known as pathogen-associated molecular patterns(PAMPs) (Jensen, S. and Thomsen, A. R. J Virol (2012) 86:2900-2910).These PAMPs include a wide array of molecular structures, such asnucleic acids, lipopolysaccharides, and glycoproteins that may bebroadly shared by multiple microbial species and are critical to theirsurvival and/or pathogenicity. Host recognition may occur by multiplepathways, such as activation of pattern recognition receptors (PRRs),which ultimately lead to downstream signaling events and culminate inthe mounting of an immune response.

To date, several PRRs have been identified that serve as sensors ofpathogenic infection. For example, the retinoic acid-inducible gene-I(RIG-I) protein is a RNA helicase that also functions as a sensor ofmicrobial-derived RNA. RIG-I is important factor in host recognition ofRNA viruses from a variety of different viral families, includingFlaviviridae (e.g., West Nile virus, Hepatitis C virus, Japaneseencephalitis virus, Dengue virus), Paramyxoviridae (e.g., Sendai virus,Newcastle disease virus, Respiratory syncytial virus, Measles virus),Rhabdoviridae (e.g., Rabies virus), Orthomyxoviridae (e.g., influenza Avirus, influenza B virus), and Arenaviridae (e.g., Lassa virus), as wellas a biomarker for the prediction of prognosis for certain types ofcancer, such as hepatocellular carcinoma (Hou, J. et al, Cancer Cell(2014) 25:49-63). The stimulator of interferon genes (STING) is acytoplasmic adaptor protein that activates the TBK1-IRF3 signalingcomplex, resulting in induction of type I interferons (IFN-β and IFN-α)and other immune pathway proteins. Other PRRs also play a role insensing microbial-derived nucleic acids, including NOD2, LGP2, MDA5, anda number of Toll-like receptors (TLRs) that are expressed on the cellsurface and within endosomal compartments.

Recent publications have highlighted the importance of RIG-I and STINGas mediators of innate and adaptive immunity, and RIG-I and STINGagonists have been recognized as immuno-oncology agents in cancertherapy (Li, X. Y. et al, Mol Cell Oncol (2014) 1:e968016; Woo, S. R.Trends in Immunol (2015) 36:250-256). In particular, RIG-I is involvedin the regulation of basic cellular processes such as hematopoieticproliferation and differentiation, maintenance of leukemic sternness,and tumorigenesis of hepatocellular carcinoma, indicating that RIG-Iperforms an essential function as a tumor suppressor. Importantly, theSTING pathway of cytosolic DNA sensing has been shown to play animportant mechanistic role in innate immune sensing, driving type I IFNproduction in cancer and in the context of immune-oncology applicationsincluding therapeutics and diagnostics.

SUMMARY OF DISCLOSURE

Cyclic dinucleotide compounds, compositions comprising cyclicdinucleotide compounds, and related methods of use are described herein.

In one aspect, the disclosure features a compound of Formula (I):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:Z is either S or O; each of B¹ and B² is independently a purinylnucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independentlyO or S; each of Y¹ and Y² is independently O, S, or NR⁵; each of L¹ andL² is independently absent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, whereineach alkyl and heteroalkyl is optionally substituted with R⁶; each of R¹and R² is independently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), or OR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀alkyl (e.g., C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl),OC(O)OC₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl, heterocyclyl, aryl,or heteroaryl, wherein each alkyl, heteroalkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R¹; R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl); R⁶ is halo,—CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; ach R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), C(O)O—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)O—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), C(O)N(R⁵)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),N(R⁵)C(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)N(R⁵)—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), O-aryl, O-heteroaryl, C(O)-aryl, C(O)-heteroaryl,OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl, C(O)O-heteroaryl, C(O)O-aryl,C(O)O-heteroaryl, C(O)N(R⁵)-aryl, C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl,N(R⁵)₂C(O)-aryl, or N(R⁵)C(O)-heteroaryl, S(O)₂N(R⁵)-aryl, wherein eachalkyl, heteroalkyl, aryl, and heteroaryl is optionally substituted byone or more R⁹; and each R⁹ is independently C₁-C₂₀ alkyl, O—C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, halo, —CN, OH, oxo, aryl, heteroaryl, O-aryl,or O-heteroaryl.

In some embodiments, the compound is a compound of Formula (I-a):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of B¹ and B² is independently a purinyl nucleobase or pyrimidinylnucleobase; each of X¹ and X² is independently O or S; each of Y¹ and Y²is independently O, S, or NR⁵; each of L¹ and L² is independentlyabsent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, wherein each alkyl andheteroalkyl is optionally substituted with R⁶; each of R¹ and R² isindependently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), orOR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀ alkyl (e.g.,C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, heteroalkyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substitutedwith one or more R¹; R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);R⁶ is halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; each R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl, C(O)O—C₁-C₂₀alkyl, OC(O)O—C₁-C₂₀ alkyl, C(O)N(R⁵)—C₁-C₂₀ alkyl, N(R⁵)C(O)—C₁-C₂₀alkyl, OC(O)N(R⁵)—C₁-C₂₀ alkyl, O-aryl, O-heteroaryl, C(O)-aryl,C(O)-heteroaryl, OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl,C(O)O-heteroaryl, C(O)O-aryl, C(O)O-heteroaryl, C(O)N(R⁵)-aryl,C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl, or N(R⁵)C(O)-heteroaryl, whereineach alkyl, heteroalkyl, aryl, and heteroaryl is optionally substitutedby one or more R⁹; and each R⁹ is independently C₁-C₂₀ alkyl, O—C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, halo, —CN, OH, oxo, aryl, heteroaryl, O-aryl,or O-heteroaryl.

In some embodiments, each of B¹ and B² is independently a purinylnucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independentlyO or S; each of Y¹ and Y² is independently O, S, or NR⁵; each of L¹ andL² is independently absent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, whereineach alkyl and heteroalkyl is optionally substituted with R⁶; each of R¹and R² is independently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), or OR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀alkyl (e.g., C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl),cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each alkyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl isoptionally substituted with 1-5 R⁸; R⁵ is hydrogen or C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl); R⁶ is halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),OR⁷, oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein eachalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionallysubstituted with 1-5 R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each₀alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionallysubstituted with 1-5 R⁹; each R⁸ is independently C₁-C₂₀ alkyl (e.g.,C₁-C₆ alkyl), C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, C(O)O-aryl,OC(O)-heteroaryl, or C(O)O-heteroaryl, wherein each alkyl, aryl, andheteroaryl is optionally substituted by 1-5 R⁹; and each R⁹ isindependently C₁-C₂₀ alkyl, O—C₁-C₂₀ alkyl, halo, —CN, OH, O—C₁-C₂₀alkyl, O—C₁-C₂₀ heteroalkyl, O-aryl, or O-heteroaryl.

In some embodiments, the compound is a compound of Formulas (I-b),(I-c), (I-d), or (I-e):

or a pharmaceutically acceptable salt thereof, wherein each of B¹, B²,X¹, X², Y¹, Y², L¹, L², R¹, R², R³, R⁴, and subvariables thereof aredefined as above.

In some embodiments, at least one of B¹ or B² is a purinyl nucleobase.In some embodiments, each of B¹ or B² is independently a purinylnucleobase. In some embodiments, B¹ is a purinyl nucleobase. In someembodiments, B² is a pyrimidinyl nucleobase. In some embodiments, B¹ isa purinyl nucleobase and B² is a pyrimidinyl nucleobase. In someembodiments, B¹ is adenosinyl or guanosinyl. In some embodiments, B² iscytosinyl, thyminyl, or uracilyl. In some embodiments, B¹ is adenosinylor guanosinyl and B² is cytosinyl, thyminyl, or uracilyl. In someembodiments, each of B¹ is and B² is independently uracilyl. In someembodiments, each of B¹ is and B² is independently adenosinyl.

In some embodiments, each of R¹ and R² is independently hydrogen, halo,or OR⁷. In some embodiments, each of R¹ and R² is independently halo(e.g., fluoro). In some embodiments, each of R¹ and R² is not hydrogenor OR⁷.

In some embodiments, X¹ is O. In some embodiments, X² is O. In someembodiments, each of X¹ and X² is independently O.

In some embodiments, Y¹ is O or S. In some embodiments, Y² is O or S. Insome embodiments, each of Y¹ and Y² is independently O or S. In someembodiments, one of Y¹ or Y² is O and the other of Y¹ or Y² is S. Insome embodiments,

each of Y¹ or Y² is independently S. In some embodiments, each of Y¹ orY² is independently O.

In some embodiments, L¹ is C₁-C₆ alkyl (e.g., CH₂). In some embodiments,L² is C₁-C₆ alkyl (e.g., CH₂). In some embodiments, each of L¹ and L² isindependently C₁-C₆ alkyl (e.g., CH₂).

In some embodiments, R³ is hydrogen, aryl, or heteroaryl, wherein aryland heteroaryl is optionally substituted with 1-5 R⁸. In someembodiments, R³ is aryl or heteroaryl, each of which is optionallysubstituted with 1-5 R⁸. In some embodiments, R³ is phenyl substitutedwith 1 R⁸.

In some embodiments, R⁴ is independently hydrogen, aryl, or heteroaryl,wherein aryl and heteroaryl is optionally substituted with 1-5 R⁸. Insome embodiments, R⁴ is aryl or heteroaryl, each of which is optionallysubstituted with 1-5 R⁸. In some embodiments, R⁴ is phenyl substitutedwith 1 R⁸.

In some embodiments, each of R³ and R⁴ is independently hydrogen, aryl,or heteroaryl, wherein aryl and heteroaryl is optionally substitutedwith 1-5 R⁸. In some embodiments, R³ is aryl or heteroaryl, each ofwhich is optionally substituted with 1-5 R⁸, and R⁴ is hydrogen. In someembodiments, R³ is phenyl substituted with 1 R⁸ and R⁴ is hydrogen. Insome embodiments, each of R³ and R⁴ is independently phenyl substitutedwith 1 R⁸.

In some embodiments, each of Y¹ and Y² is O and each of R³ and R⁴ isindependently hydrogen. In some embodiments, Y² is O and R⁴ is hydrogen.In some embodiments, each of Y¹ and Y² is independently S and each of R³and R⁴ is independently substituted with 1 R⁸. In some embodiments, Y¹is S and R³ is substituted with 1 R⁸.

In some embodiments, each R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl,OC(O)O—C₁-C₂₀ alkyl, OC(O)N(R⁵)—C₁-C₂₀ alkyl, O-aryl, C(O)-aryl,OC(O)-aryl, or C(O)N(R⁵)-aryl, wherein each alkyl, heteroalkyl, aryl,and heteroaryl is optionally substituted by one or more R⁹.

In some embodiments, R⁸ is OC(O)-aryl optionally substituted by 1-5 R⁹(e.g., 1 R⁹).

In some embodiments, R⁹ is O—C₁-C₁₂ alkyl (e.g., O—CH₂(CH₂)₈CH₃). Insome embodiments, R⁹ is O—C₁-C₁₀ alkyl (e.g., O—CH₂(CH₂)₈CH₃). In someembodiments, R⁹ is O—C₁-C₈ alkyl (e.g., O—CH₂(CH₂)₆CH₃). In someembodiments, R⁹ is O—C₁-C₆ alkyl (e.g., O—CH₂(CH₂)₄CH₃).

In some embodiments, the compound is a compound of Formula (I-f):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of B¹ and B² is independently a purinyl nucleobase or pyrimidinylnucleobase; each of X¹ and X² is independently O or S; each of Y¹ and Y²is independently O, S, or NR⁵; each of L¹ and L² is independentlyabsent, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl) or C₁-C₂₀ heteroalkyl (e.g.,C₁-C₆ heteroalkyl), wherein each C₁-C₂₀ alkyl and C₁-C₂₀ heteroalkyl isoptionally substituted with R⁶;each of R¹ and R² is independently halo; each of R³ and R⁴ isindependently hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), C₁-C₂₀heteroalkyl (e.g., C₁-C₆ heteroalkyl), cycloalkyl, heterocyclyl, aryl,or heteroaryl, wherein each C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substitutedwith 1-5 R⁸; R⁵ is hydrogen or C₁-C₆ alkyl; R⁶ is halo, —CN, C₁-C₂₀alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl, heterocyclyl, aryl, orheteroaryl, wherein each C₁-C₂₀ alkyl, cycloalkyl, heterocyclyl, aryl,or heteroaryl is optionally substituted with 1-5 R⁹; R⁷ is hydrogen,C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl, heterocyclyl, aryl, orheteroaryl, wherein each C₁-C₂₀ alkyl, cycloalkyl, heterocyclyl, aryl,or heteroaryl is optionally substituted with 1-5 R⁹; each R⁸ isindependently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), C(O)-aryl,C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, wherein each C₁-C₂₀alkyl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl isoptionally substituted by 1-5 R⁹; and each R⁹ is independently C₁-C₂₀alkyl, halo, —CN, OH, O—C₁-C₂₀ alkyl, O—C₁-C₂₀heteroalkyl, O-aryl, orO-heteroaryl.

In some embodiments, the compound is a compound of Formula (I-g):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of B¹ and B² is independently a purinyl nucleobase or pyrimidinylnucleobase; each of X¹ and X² is independently O; each of Y¹ and Y² isindependently O or S; each of L¹ and L² is independently absent or C₁-C₆alkyl; each of R¹ and R² is independently halo or OH; each of R³ and R⁴is independently hydrogen or aryl optionally substituted with 1-5 R⁸;each R⁸ is independently OC(O)-aryl optionally substituted by 1-5 R⁹;and each R⁹ is independently O—C₁-C₁₂ alkyl.

In some embodiments, the compound of Formula (I) is selected from acompound of Table 1, Table 2, or a pharmaceutically acceptable saltthereof.

In some embodiments, the compound of Formula (I-a) is selected from acompound of Table 1, Table 2, or a pharmaceutically acceptable saltthereof.

In another aspect, the disclosure features a method of treating cancerin a subject, the method comprising administering to the subject acompound of Formula (I),

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:Z is either S or O; each of B¹ and B² is independently a purinylnucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independentlyO or S; each of Y¹ and Y² is independently O, S, or NR⁵; each of L¹ andL² is independently absent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, whereineach alkyl and heteroalkyl is optionally substituted with R⁶; each of R¹and R² is independently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), or OR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀alkyl (e.g., C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl),OC(O)OC₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl, heterocyclyl, aryl,or heteroaryl, wherein each alkyl, heteroalkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R¹; R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl); R⁶ is halo,—CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; ach R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), C(O)O—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)O—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), C(O)N(R⁵)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),N(R⁵)C(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)N(R⁵)—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), O-aryl, O-heteroaryl, C(O)-aryl, C(O)-heteroaryl,OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl, C(O)O-heteroaryl, C(O)O-aryl,C(O)O-heteroaryl, C(O)N(R⁵)-aryl, C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl,N(R⁵)₂C(O)-aryl, or N(R⁵)C(O)-heteroaryl, S(O)₂N(R⁵)-aryl, wherein eachalkyl, heteroalkyl, aryl, and heteroaryl is optionally substituted byone or more R⁹; and each R⁹ is independently C₁-C₂₀ alkyl, O—C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, halo, —CN, OH, oxo, aryl, heteroaryl, O-aryl,or O-heteroaryl.

In some embodiments, the disclosure features a method of treating cancerin a subject, the method comprising administering to the subject acompound of Formula (I-a),

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of B¹ and B² is independently a purinyl nucleobase or pyrimidinylnucleobase; each of X¹ and X² is independently O or S; each of Y¹ and Y²is independently O, S, or NR⁵; each of L¹ and L² is independentlyabsent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, wherein each alkyl andheteroalkyl is optionally substituted with R⁶; each of R¹ and R² isindependently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), orOR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀ alkyl (e.g.,C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, heteroalkyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substitutedwith one or more R¹; R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);R⁶ is halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; each R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl, C(O)O—C₁-C₂₀alkyl, OC(O)O—C₁-C₂₀ alkyl, C(O)N(R⁵)—C₁-C₂₀ alkyl, N(R⁵)C(O)—C₁-C₂₀alkyl, OC(O)N(R⁵)—C₁-C₂₀ alkyl, O-aryl, O-heteroaryl, C(O)-aryl,C(O)-heteroaryl, OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl,C(O)O-heteroaryl, C(O)O-aryl, C(O)O-heteroaryl, C(O)N(R⁵)-aryl,C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl, or N(R⁵)C(O)-heteroaryl, whereineach alkyl, heteroalkyl, aryl, and heteroaryl is optionally substitutedby one or more R⁹; and each R⁹ is independently C₁-C₂₀ alkyl, O—C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, halo, —CN, OH, oxo, aryl, heteroaryl, O-aryl,or O-heteroaryl.

In some embodiments, the cancer is a cancer of the breast, bone, brain,cervix, colon, gastrointestinal tract, eye, gall bladder, lymph nodes,blood, lung, liver, skin, mouth, prostate, ovary, penis, pancreas,uterus, testicles, stomach, thymus, thyroid, or other part of the body(e.g., a cancer of the liver). In some embodiments, the cancer hasdifferential expression of STING relative to the noncancerous tissue,e.g., liver cancer, melanoma, skin cancer, or thyroid cancer.

In some embodiments, the cancer comprises a PD-1 resistant tumor.

In some embodiments, the method comprises oral administration of thecompound of Formula (I) or a pharmaceutically acceptable salt thereof,or a pharmaceutical composition thereof. In some embodiments, the methodcomprises oral administration of the compound of Formula (I-a) or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof. In some embodiments, the method comprisesparenteral administration (e.g., subcutaneous, intramuscular,intraperitoneal, or intravenous administration) of the compound ofFormula (I) or a pharmaceutically acceptable salt thereof, or apharmaceutical composition thereof. In some embodiments, the methodcomprises parenteral administration (e.g., subcutaneous, intramuscular,intraperitoneal, or intravenous administration) of the compound ofFormula (I-a) or a pharmaceutically acceptable salt thereof, or apharmaceutical composition thereof. In some embodiments, the methodcomprises intraperitoneal administration of the compound of Formula (I)or a pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof. In some embodiments, the method comprisesintraperitoneal administration of the compound of Formula (I-a) or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof. In some embodiments, the method comprisesintratumoral administration of the compound of Formula (I) or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof. In some embodiments, the method comprisesintratumoral administration of the compound of Formula (I-a) or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof.

In some embodiments, the method further comprises administration of anadditional agent (e.g., an anticancer agent or an immunooncology agent).In some embodiments, the additional agent comprises methotrexate,5-fluorouracil, doxorubicin, vincristine, bleomycin, vinblastine,dacarbazine, toposide, cisplatin, epirubicin, or sorafenib tosylate.

In another aspect, the disclosure features a composition comprising avaccine, and a vaccine adjuvant comprising a compound of Formula (I),

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:Z is either S or O; each of B¹ and B² is independently a purinylnucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independentlyO or S; each of Y¹ and Y² is independently O, S, or NR⁵; each of L¹ andL² is independently absent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, whereineach alkyl and heteroalkyl is optionally substituted with R⁶; each of R¹and R² is independently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), or OR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀alkyl (e.g., C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl),OC(O)OC₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl, heterocyclyl, aryl,or heteroaryl, wherein each alkyl, heteroalkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R¹; R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl); R⁶ is halo,—CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; ach R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), C(O)O—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)O—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), C(O)N(R⁵)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),N(R⁵)C(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)N(R⁵)—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), O-aryl, O-heteroaryl, C(O)-aryl, C(O)-heteroaryl,OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl, C(O)O-heteroaryl, C(O)O-aryl,C(O)O-heteroaryl, C(O)N(R⁵)-aryl, C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl,N(R⁵)₂C(O)-aryl, or N(R⁵)C(O)-heteroaryl, S(O)₂N(R⁵)-aryl, wherein eachalkyl, heteroalkyl, aryl, and heteroaryl is optionally substituted byone or more R⁹; and each R⁹ is independently C₁-C₂₀ alkyl, O—C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, halo, —CN, OH, oxo, aryl, heteroaryl, O-aryl,or O-heteroaryl.

In some embodiments, the disclosure features a composition comprising avaccine, and a vaccine adjuvant comprising a compound of Formula (I-a),

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of B¹ and B² is independently a purinyl nucleobase or pyrimidinylnucleobase; each of X¹ and X² is independently O or S; each of Y¹ and Y²is independently O, S, or NR⁵; each of L¹ and L² is independentlyabsent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, wherein each alkyl andheteroalkyl is optionally substituted with R⁶; each of R¹ and R² isindependently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), orOR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀ alkyl (e.g.,C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, heteroalkyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substitutedwith one or more R¹; R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);R⁶ is halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; each R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl, C(O)O—C₁-C₂₀alkyl, OC(O)O—C₁-C₂₀ alkyl, C(O)N(R⁵)—C₁-C₂₀ alkyl, N(R⁵)C(O)—C₁-C₂₀alkyl, OC(O)N(R⁵)—C₁-C₂₀ alkyl, O-aryl, O-heteroaryl, C(O)-aryl,C(O)-heteroaryl, OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl,C(O)O-heteroaryl, C(O)O-aryl, C(O)O-heteroaryl, C(O)N(R⁵)-aryl,C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl, or N(R⁵)C(O)-heteroaryl, whereineach alkyl, heteroalkyl, aryl, and heteroaryl is optionally substitutedby one or more R⁹; each R⁹ is independently C₁-C₂₀ alkyl, O—C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, halo, —CN, OH, oxo, aryl, heteroaryl, O-aryl,or O-heteroaryl, wherein each alkyl, heteroalkyl, aryl, or heteroaryl isoptionally substituted with one or more R¹⁰; each R¹⁰ is independentlyC₁-C₂₀ alkyl, C₁-C₂₀ alkenyl, C₁-C₂₀ alkynyl, C₁-C₂₀ heteroalkyl, halo,—CN, or OH, oxo; and each R⁵ is independently hydrogen or C₁-C₂₀ alkyl.

In another aspect, the disclosure features a method of inducing theexpression of a pattern recognition receptors (PR) for immune-modulationin a subject, the method comprising administering to the subject acompound of Formula (I),

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:Z is either S or O; each of B¹ and B² is independently a purinylnucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independentlyO or S; each of Y¹ and Y² is independently O, S, or NR⁵; each of L¹ andL² is independently absent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, whereineach alkyl and heteroalkyl is optionally substituted with R⁶; each of R¹and R² is independently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), or OR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀alkyl (e.g., C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl),OC(O)OC₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl, heterocyclyl, aryl,or heteroaryl, wherein each alkyl, heteroalkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R¹; R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl); R⁶ is halo,—CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; ach R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), C(O)O—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)O—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), C(O)N(R⁵)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),N(R⁵)C(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)N(R⁵)—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), O-aryl, O-heteroaryl, C(O)-aryl, C(O)-heteroaryl,OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl, C(O)O-heteroaryl, C(O)O-aryl,C(O)O-heteroaryl, C(O)N(R⁵)-aryl, C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl,N(R⁵)₂C(O)-aryl, or N(R⁵)C(O)-heteroaryl, S(O)₂N(R⁵)-aryl, wherein eachalkyl, heteroalkyl, aryl, and heteroaryl is optionally substituted byone or more R⁹; and each R⁹ is independently C₁-C₂₀ alkyl, O—C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, halo, —CN, OH, oxo, aryl, heteroaryl, O-aryl,or O-heteroaryl.

In some embodiments, the disclosure features a method of inducing theexpression of a pattern recognition receptors (PRR) forimmune-modulation in a subject, the method comprising administering tothe subject a compound of Formula (I-a),

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of B¹ and B² is independently a purinyl nucleobase or pyrimidinylnucleobase; each of X¹ and X² is independently O or S; each of Y¹ and Y²is independently O, S, or NR⁵; each of L¹ and L² is independentlyabsent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, wherein each alkyl andheteroalkyl is optionally substituted with R⁶; each of R¹ and R² isindependently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), orOR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀ alkyl (e.g.,C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, heteroalkyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substitutedwith one or more R¹; R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);R⁶ is halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; each R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl, C(O)O—C₁-C₂₀alkyl, OC(O)O—C₁-C₂₀ alkyl, C(O)N(R⁵)—C₁-C₂₀ alkyl, N(R⁵)C(O)—C₁-C₂₀alkyl, OC(O)N(R⁵)—C₁-C₂₀ alkyl, O-aryl, O-heteroaryl, C(O)-aryl,C(O)-heteroaryl, OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl,C(O)O-heteroaryl, C(O)O-aryl, C(O)O-heteroaryl, C(O)N(R⁵)-aryl,C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl, or N(R⁵)C(O)-heteroaryl, whereineach alkyl, heteroalkyl, aryl, and heteroaryl is optionally substitutedby one or more R⁹; each R⁹ is independently C₁-C₂₀ alkyl, O—C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, halo, —CN, OH, oxo, aryl, heteroaryl, O-aryl,or O-heteroaryl.

In another aspect, the disclosure features a method of inducing theexpression of a pattern recognition receptor (PRR) for immunomodulationand inducing a therapeutic response in a subject having cancer, themethod comprising administering to the subject a compound of Formula(I),

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:Z is either S or O; each of B¹ and B² is independently a purinylnucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independentlyO or S; each of Y¹ and Y² is independently O, S, or NR⁵; each of L¹ andL² is independently absent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, whereineach alkyl and heteroalkyl is optionally substituted with R⁶; each of R¹and R² is independently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), or OR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀alkyl (e.g., C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl),OC(O)OC₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl, heterocyclyl, aryl,or heteroaryl, wherein each alkyl, heteroalkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R¹; R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl); R⁶ is halo,—CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; ach R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), C(O)O—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)O—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), C(O)N(R⁵)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),N(R⁵)C(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)N(R⁵)—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), O-aryl, O-heteroaryl, C(O)-aryl, C(O)-heteroaryl,OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl, C(O)O-heteroaryl, C(O)O-aryl,C(O)O-heteroaryl, C(O)N(R⁵)-aryl, C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl,N(R⁵)₂C(O)-aryl, or N(R⁵)C(O)-heteroaryl, S(O)₂N(R⁵)-aryl, wherein eachalkyl, heteroalkyl, aryl, and heteroaryl is optionally substituted byone or more R⁹; and each R⁹ is independently C₁-C₂₀ alkyl, O—C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, O—C₁-C₂₀—NR¹⁰R¹⁰, halo, —CN, OH, oxo, aryl,heteroaryl, O-aryl, or O-heteroaryl.

In some embodiments, the disclosure features a method of inducing theexpression of a pattern recognition receptor (PRR) for immunomodulationand inducing a therapeutic response in a subject having cancer, themethod comprising administering to the subject a compound of Formula(I-a),

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of B¹ and B² is independently a purinyl nucleobase or pyrimidinylnucleobase; each of X¹ and X² is independently O or S; each of Y¹ and Y²is independently O, S, or NR⁵; each of L¹ and L² is independentlyabsent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, wherein each alkyl andheteroalkyl is optionally substituted with R⁶; each of R¹ and R² isindependently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), orOR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀ alkyl (e.g.,C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, heteroalkyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substitutedwith one or more R¹; R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);R⁶ is halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; each R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl, C(O)O—C₁-C₂₀alkyl, OC(O)O—C₁-C₂₀ alkyl, C(O)N(R⁵)—C₁-C₂₀ alkyl, N(R⁵)C(O)—C₁-C₂₀alkyl, OC(O)N(R⁵)—C₁-C₂₀ alkyl, O-aryl, O-heteroaryl, C(O)-aryl,C(O)-heteroaryl, OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl,C(O)O-heteroaryl, C(O)O-aryl, C(O)O-heteroaryl, C(O)N(R⁵)-aryl,C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl, or N(R⁵)C(O)-heteroaryl, whereineach alkyl, heteroalkyl, aryl, and heteroaryl is optionally substitutedby one or more R⁹; each R⁹ is independently C₁-C₂₀ alkyl, O—C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, halo, —CN, OH, oxo, aryl, heteroaryl, O-aryl,or O-heteroaryl.

In another aspect, the present disclosure features a method of inducingan immune response in a subject, the method comprising administering tothe subject a compound of Formula (I),

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:Z is either S or O; each of B¹ and B² is independently a purinylnucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independentlyO or S; each of Y¹ and Y² is independently O, S, or NR⁵; each of L¹ andL² is independently absent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, whereineach alkyl and heteroalkyl is optionally substituted with R⁶; each of R¹and R² is independently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), or OR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀alkyl (e.g., C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl),OC(O)OC₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl, heterocyclyl, aryl,or heteroaryl, wherein each alkyl, heteroalkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R¹; R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl); R⁶ is halo,—CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; ach R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), C(O)O—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)O—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), C(O)N(R⁵)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),N(R⁵)C(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)N(R⁵)—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), O-aryl, O-heteroaryl, C(O)-aryl, C(O)-heteroaryl,OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl, C(O)O-heteroaryl, C(O)O-aryl,C(O)O-heteroaryl, C(O)N(R⁵)-aryl, C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl,N(R⁵)₂C(O)-aryl, or N(R⁵)C(O)-heteroaryl, S(O)₂N(R⁵)-aryl, wherein eachalkyl, heteroalkyl, aryl, and heteroaryl is optionally substituted byone or more R⁹; and each R⁹ is independently C₁-C₂₀ alkyl, O—C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, halo, —CN, OH, oxo, aryl, heteroaryl, O-aryl,or O-heteroaryl.

In some embodiments aspect, the present disclosure features a method ofinducing an immune response in a subject, the method comprisingadministering to the subject a compound of Formula (I-a),

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of B¹ and B² is independently a purinyl nucleobase or pyrimidinylnucleobase; each of X¹ and X² is independently O or S; each of Y¹ and Y²is independently O, S, or NR⁵; each of L¹ and L² is independentlyabsent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, wherein each alkyl andheteroalkyl is optionally substituted with R⁶; each of R¹ and R² isindependently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), orOR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀ alkyl (e.g.,C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, heteroalkyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substitutedwith one or more R¹; R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);R⁶ is halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; each R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl, C(O)O—C₁-C₂₀alkyl, OC(O)O—C₁-C₂₀ alkyl, C(O)N(R⁵)—C₁-C₂₀ alkyl, N(R⁵)C(O)—C₁-C₂₀alkyl, OC(O)N(R⁵)—C₁-C₂₀ alkyl, O-aryl, O-heteroaryl, C(O)-aryl,C(O)-heteroaryl, OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl,C(O)O-heteroaryl, C(O)O-aryl, C(O)O-heteroaryl, C(O)N(R⁵)-aryl,C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl, or N(R⁵)C(O)-heteroaryl, whereineach alkyl, heteroalkyl, aryl, and heteroaryl is optionally substitutedby one or more R⁹; each R⁹ is independently C₁-C₂₀ alkyl, O—C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, halo, —CN, OH, oxo, aryl, heteroaryl, O-aryl,or O-heteroaryl.

In some embodiments, the immune response comprises antitumoral immunity.In some embodiments, the immune response comprises induction of a PRR(e.g., STING, RIG-I, MDA5).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show that an exemplary compound engages/bind STING toactivate type I IFN signaling. FIG. 1A describes the results of aprimary screen for STING agonists, in which HEK293 cells stablyexpressing ISG54 (ISRE)-promoter driven firefly luciferase gene wereused to screen a compound library. Cells transfected with human STINGand internal control Renilla-luciferase were treated with 25 uMexemplary compounds, and TRF activity was assessed by measuringluciferase levels.

FIGS. 2A-2F show the potency comparison of an exemplary compound (Cmd 1)vs. a natural STING ligand, 2′-3′ cGAMP.

FIGS. 3A-3B show that an exemplary compound has STING-dependentactivity.

FIG. 4 shows IRF induction by exemplary compounds.

FIGS. 5A-5B show that exemplary compounds engage with STING and activateSTING-dependent type I IFN and NF-κB signaling in HEK293 cells.

FIG. 6 shows NF-KB induction by exemplary compounds.

FIGS. 7A-7E show that an exemplary compound causes cell death byapoptosis through the modulation of BAX and BCL-2 levels.

FIGS. 8A-8B show the selective induction of apoptosis by Cmd 1 in acutemonocytic leukemia cell line (THP1) vs. PBMCs.

FIGS. 9A-9B show that an exemplary compound (Cmd 1) causes selective andenhanced induction of ISG and PRR-associated genes in acute monocyticleukemia cell line (THP1) compared to primary cells PBMCs. Geneexpression analysis was conducted in THP1 and PBMCs.

FIGS. 10A-10B show that an exemplary compound inhibits tumor cellgrowth.

FIGS. 11A-11B show that an exemplary compound has STING-dependent IRFactivity but does not cause NF-kB induction.

FIG. 12 shows that an exemplary compound activates TRF signaling in THP1cells.

FIGS. 13A-13D show that an exemplary compound has similar activity asnatural STING ligand 2′-3′ cGAMP.

FIG. 14 shows that an exemplary compound directly binds to STING.

FIGS. 15A-15B show that an exemplary compound has STING dependent IRFactivity but does not cause NF-kB induction.

FIG. 16 shows that an exemplary compound directly binds to STING.

FIGS. 17A-17B show that an exemplary compound has STING dependentactivity.

FIGS. 18A-18D show that an exemplary compound has similar potency asnatural STING ligand 2′-3′ cGAMP.

FIGS. 19A-19B shows that an exemplary compound has enhanced activity inacute monocytic leukemia cell line (THP1) compared to primary cellsPBMCs.

FIGS. 20A-20C show IRF induction by an exemplary compound.

FIGS. 21A-21C show IRF induction by an exemplary compound.

FIGS. 22A-22B are graphs showing the evaluation of percent (%) IRFinduction (FIG. 22A) and percent (%) NF-κB (FIG. 22B) by Cmd 1, Cmd 1A,and Cmd 1B in THP1 dual cells that carry both the secreted embryonicalkaline phosphatase (SEAP) reporter gene under the control of an IFN-βminimal promoter fused to five copies of the NF-κB consensustranscription response element and Lucia reporter gene under the controlof an ISG54 minimal promoter.

FIGS. 23A-23D are graphs showing the induction of IRF (FIGS. 23A-23B)and NF-κB (FIGS. 23C-23D) by Cmd 1, and indicate that Cmd1 is taken upby cells without the use of transfection agents.

FIGS. 24A-24B are graphs showing the induction of IRF by Cmd 3, andindicate that Cmd 3 is taken up by cells without the use of transfectionagents.

FIGS. 25A-25D are graphs showing the induction of IRF (FIGS. 25A-25B)and NF-κB (FIGS. 25C-25D) by Cmd 12, and indicate that Cmd12 is taken upby cells without the use of transfection agents.

FIGS. 26A-26D are graphs showing the induction of IRF (FIGS. 26A-26B)and NF-κB (FIGS. 26C-26D) by Cmd 13, and indicate that Cmd13 is taken upby cells without the use of transfection agents.

FIGS. 27A-27D are graphs showing the induction of IRF (FIGS. 27A-27B)and NF-κB (FIGS. 27C-27D) by Cmd 14, and indicate that Cmd14 is taken upby cells without the use of transfection agents.

FIGS. 28A-28D are graphs showing the induction of IRF (FIGS. 28A-28B)and NF-κB (FIGS. 28C-28D) by Cmd 15, and indicate that Cmd15 is taken upby cells without the use of transfection agents.

FIGS. 29A-29B are charts comparing the relative induction of IRF (FIG.29A) and NF-κB (FIG. 291B) by Cmd 1, Cmd 3, Cmd 12, Cmd 13, Cmd 14, andCmd 15.

FIGS. 30A-30B are graphs showing the stability of Cmd 1 in serum (FIG.30A) and in microsomes (FIG. 30B). In FIG. 30B, Peak 1 and Peak 2represent Cmds 1-A and 1-B, respectively.

FIGS. 31A-31B are graphs showing the stability of Cmd 15 in serum (FIG.31A) and in microsomes (FIG. 31B). In FIG. 31B, Peak 1 and Peak 2represent Cmds 15-A and 15-B, respectively.

FIGS. 32A-32B are charts comparing the induction of IRF (FIG. 32A) andNF-κB (FIG. 32B) by Cmd 15 and its isomers, Cmd 15-A and Cmd 15-B.

FIG. 33 is a chart showing the induction of apoptosis through %cytoxicity of THP1 cells by Cmd 15 and its isomers, Cmd 15-A and Cmd15-B.

FIGS. 34A-34B show that Cmd 1 binds to STING to activate type 1 IFNsignaling, similar to 2′,3′-cGAMP.

FIG. 35 is a chart showing that Cmd 1 is highly active in mousemacrophages in activating type 1 IFN signaling, similar to 2′,3′-cGAMP.

FIGS. 36A-36B are graphs that show that Cmd 1, Cmd 5, Cmd 12, Cmd 13,Cmd 14, and Cmd 15 are more active against the natural STING ligand3′,3′-cGAMP in human monocytes (FIG. 36A) and mouse macrophages (FIG.36B).

FIGS. 37A-37B are graphs that show the induction of type I IFN signalingin HEK293 (FIG. 37A) and THP1 (FIG. 37B) cells by Cmd 1 and its isomersCmd 1A and Cmd 1B.

FIGS. 38A-38B are charts showing that Cmd 1 and Cmd 15 induce type IIIinterferon (IL-29) production in THP1 cells (FIG. 38A), and that bothCmd 1 and Cmd 15 are taken up by cells without use of a transfectionreagent (FIG. 38B).

FIGS. 39A-39B are graphs comparing the induction of type I IFN signalingin THP1 cells by Cmd 1, Cmd 13, Cmd 15 as STING agonists.

FIGS. 40A-40B are charts comparing the induction of IRF (FIG. 42A) andNF-κB (FIG. 42B) by Cmd 15 and Cmd 16.

FIGS. 41A-41B show that Cmd1 is capable of activating the majorSTING-HAQ polymorphic variant in humans.

FIG. 42 shows that residues R238 and Y167 in STING Laboratory-generatedloss-of-function STING mutants (STING-R238A and STING-Y167A) arecritical for Cmd1 as well as cGAMP activation of STING-dependent IFNresponse.

FIG. 43 shows IRF-type I IFN activity by Cmd1 in co-cultured tumor/THP1cell system.

FIGS. 44A-44B show that Cmd1 inhibits tumor cell growth in tumor cellsand THP1 cells using high-content image-based approach and is STINGdependent.

FIGS. 45A-45B show that Cmd 1 causes apoptosis acute monocytic leukemiacells.

FIGS. 46A-46E show that Cmd 1 induces apoptosis in the mouse lymphomacell line A20.

FIGS. 47A-47B show that Cmd1 causes apoptosis of mouse melanoma cells.

FIGS. 48A-48D show that Cmd1 inhibits mouse A20 B cell lymphoma tumorcells.

FIG. 49 shows anti-tumor activity of Cmd1 using high-content image-basedapproach.

FIGS. 50A-50F show that the induction of cell death by Cmd1 isSTING-mediated.

FIGS. 51A-51C show the results of a gel shift assay indicating that Cmd1 binds to STING. A close structural analog of Cmd 1 carrying afluorescent substituent was synthesized for Gel Shift Assay. FIG. 51Ashows 250 μM of Cmd 1 analog with 20 μM to 0 μM of STING. FIG. 51B shows10 μM of STING with 1 mM to 0 mM of Cmd 1 analog. FIG. 51C shows animmunoblot to detect STING.

FIGS. 52A-52P show analysis of IRF3 & NF-kB pathways after Cmd 1treatment: FIGS. 52A-52P show immunoblots in which THP-1 cells weretreated with 5 μM Cmd 1 or 2′-3′ cGAMP.

FIGS. 53A-53C show images in which THP-1 derived macrophages weretreated with Cmd 1 or DMSO control for 2 hrs (FIG. 53A), 4 hrs (FIG.53B), or 6 hrs (FIG. 53C) and analyzed for nuclear translocation. Cellswere imaged on IXM (Molecular Devices) (40×) and were analyzed usingImageJ.

FIGS. 54A-54B show the evaluation of IFN secretion and gene expressionafter Cmd 1 treatment. FIG. 54A is a graph showing the fold induction ofgene expression in THP-1 cells treated with 5 uM of either Cmd 1 or2′3-cGAMP. Gene expression was evaluated by Taqman Assays. FoldInduction was calculated by ΔΔct method. In FIG. 54B, THP-1 cells weretreated with 1 uM of Cmd 1 and secretion of certain cytokines wasevaluated by on Quansys Biosciences' (Logan, Utah) Q-Plex™ Human Custom,IFN, and IL-1 Family multiplexed ELISA arrays.

FIG. 54C-54D show the induction of apoptosis-related genes and ISGs byan exemplary compound (Cmd 1) compared with 2′3′-cGAMP in A20 mouse Bcell lymphoma tumor cells. In FIG. 54D, a higher BAX/BCL2 ratio in cellsadministered Cmd 1 promotes apoptosis via upregulation of caspase 3.

FIGS. 55A-55G are graphs showing the induction of various cytokines byCmd 1 in wild type THP1 cells as determined by multiplex ELISA.

FIGS. 56A-56D are graphs showing that an exemplary compound (Cmd 1)strongly activates the IRF-type I and type III IFN response.

FIG. 57 is a chart showing that an exemplary compound (Cmd 1) activateshuman natural killer (NK) cells and induces IFN-γ production.

FIGS. 58A-58B show that an exemplary compound (Cmd 1) potently inhibitslymphoma tumor growth in the syngeneic A20 lymphoma model.

FIGS. 59A-59D are graphs showing that an exemplary compound (Cmd 1)administered in combination with cyclophosphamide results in tumor-freesurvival in a syngeneic A20 lymphoma mouse model.

FIGS. 60A-60B show that monotherapy of Cmd 1 and combination therapy ofCmd 1 plus cyclophosphamide significantly improve the survival rate ofmice in the syngeneic A20 lymphoma mouse model. Note that in FIG. 60B,VS1 refers to Cmd 1.

FIGS. 61A-61D are images showing immunohistochemistry data on tissuestaken from mice treated with Cmd 1. The images show that the anti-tumoractivity of Cmd 1 correlates with the induction of the innate andadaptive immune response.

FIGS. 62A-62B show that an exemplary compound (Cmd 1) is highlyeffective in inhibiting tumor growth in the syngeneic CT26 colon cancermodel.

FIGS. 63A-63B show that monotherapy of Cmd 1 and combination therapy ofCmd 1 plus an anti-CTLA4 antibody significantly improve the survivalrate of mice in the syngeneic CT26 colon cancer mouse model. Note thatin FIG. 63B, VS1 refers to Cmd 1.

FIG. 64 shows that mice that are found to be tumor-free followingtreatment with either Cmd 1 or Cmd1+cyclocphosphamide experience notumor growth compared with control upon re-challenging the mice withtumor cells (A20 lymphoma tumor challenge study).

FIGS. 65A-65H are images showing immunohistochemistry data using ananti-CD38 antibody on tumor tissue collected from mice treated withvehicle (FIGS. 65A-65D) or Cmd 1 (FIGS. 65E-65H) in the syngeneic A20lymphoma model. The images show that Cmd 1 induces migration of CD8 Tinto the tumor site.

FIGS. 66A-66H are images showing immunohistochemistry data using ananti-granzyme B antibody on tumor tissue collected from mice treatedwith vehicle (FIGS. 66A-66D) or Cmd 1 (FIGS. 66E-66H) in the syngeneicA20 lymphoma model. The images show that Cmd 1 induces migration of NKcells into the tumor site.

FIGS. 67A-67H are images showing immunohistochemistry data using ananti-F4/80 antibody on tumor tissue collected from mice treated withvehicle (FIGS. 67A-67D) or Cmd 1 (FIGS. 67E-67H) in the syngeneic A20lymphoma model. The images show that Cmd 1 induces migration ofmacrophages into the tumor site.

FIGS. 68A-68G show administration of Cmd 1 to a panel of normal celllines, indicating that Cmd 1 is non-cytotoxic.

FIGS. 69A-69D show that palmitoylation of STING is involved in Cmd1-induced activation of NF-κB (FIGS. 69A-69B) and the IRF-type Iinterferon response in THP1 cells (FIGS. 69C-69D).

FIG. 70 is a graph showing that intraperitoneal administration of Cmd 1causes significant decline in tumor volume in the syngeneic mousemetastatic breast cancer model as described in Example 12.

FIG. 71 is a graph showing the results of the oral dosage study,indicating that all participating subjects are within acceptable bodyweight ranges.

FIG. 72 is a graph showing the anti-tumor activity of Cmd 1, Cmd X, andCmd 21 in the syngeneic mouse A20 lymphoma model. All compounds showedconsiderable tumor growth inhibition compared with the vehicle.

FIGS. 73A-73B are graphs showing the abscopal antitumoral activity ofCmd 1 when administered intratumorally in CT26 colon cancer model. FIG.73A shows tumor volume of tumor in left flank and FIG. 73B shows tumorvolume of tumor on right flank over 13 days post initiation treatment.Cmd 1 showed considerable tumor growth inhibition compared with thevehicle.

FIG. 74 is a graph showing effects on tumor growth at a dose of vehicleand 10 μg, 30 μg, and 100 μg of Cmd 1 in a CT26 colon cancer model. Cmd1 showed considerable tumor growth inhibition at all three dosescompared with the vehicle.

FIG. 75 is a graph showing effects on tumor growth of Cmd 1 and vehiclein a 4T1 breast cancer model. Cmd 1 showed considerable tumor growthinhibition compared with the vehicle.

FIGS. 76A-76D are bar graphs percent induction of CD8+ T cells, CD4+ Tcells, and MDSCs by Cmd 1 in spleen, lymph nodes and blood on day 19measured by flow cytometry. Cmd 1 showed increase in CD8+ T cells, CD4+T cells, and MDSCs when compared with the vehicle.

FIG. 77 is a graph showing anti-tumor activity of vehicle Cmd 1, Cmd 1A(isomer of Cmd 1), and Cmd 21. The graph shows that Cmd 1, Cmd 1A andCmd 21 inhibit mouse A20 B cell lymphoma tumor cells.

FIG. 78 is a Kaplan-Meier plot showing that Cmd 1, Cmd 1A, and Cmd 21significantly improve the survival rate of mice in the A26 lymphomamodel.

FIG. 79 is a graph showing effects on tumor growth of intratumoraladministration of Cmd 1, Cmd 21, and Cmd 25 in a CT26 colorectalcarcinoma model. Cmd 1, Cmd 21, and Cmd 25 showed considerable tumorgrowth inhibition compared with the vehicle.

FIG. 80 is a graph showing effects on tumor growth of intratumoraladministration of vehicle, vehicle and Ethanol. Cmd 1, Cmd 1A, and Cmd1A in a CT26 colorectal carcinoma model. Cmd 1, Cmd 21, and Cmd 25showed considerable tumor growth inhibition compared with the vehicle.

FIG. 81 is a graph showing the stability of Cmd 1 in Rabbit serum (FIG.81A) and in Human microsomes (FIG. 81B).

FIG. 82 are luminescence images showing effects on tumor growth ofintraperitoneal administration of Cmd 1 in a 4T1 breast cancer syngeneicmouse model. Cmd 1 showed considerable tumor growth inhibition at allthree doses compared with the vehicle.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to methods of activating and/or inducingthe expression of PRRs (e.g., STING) in a subject, in particular for thetreatment of a proliferative disease (e.g., cancer). In someembodiments, the method comprises administration of a compound ofFormula (I) or pharmaceutically acceptable salt thereof. It is to benoted that induction of any PRR with these compounds can stimulateinterferon and/or NF-KB production which can induce the expression of avariety of PRRs which are inducible genes by feedback mechanism.

Definitions

As used herein, the articles “a” and “an” refer to one or to more thanone (e.g., to at least one) of the grammatical object of the article.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values.

As used herein, the term “acquire” or “acquiring” as the terms are usedherein, refer to obtaining possession of a physical entity (e.g., asample, e.g., blood sample or liver biopsy specimen), or a value, e.g.,a numerical value, by “directly acquiring” or “indirectly acquiring” thephysical entity or value. “Directly acquiring” means performing aprocess (e.g., an analytical method) to obtain the physical entity orvalue. “Indirectly acquiring” refers to receiving the physical entity orvalue from another party or source (e.g., a third party laboratory thatdirectly acquired the physical entity or value). Directly acquiring avalue includes performing a process that includes a physical change in asample or another substance, e.g., performing an analytical processwhich includes a physical change in a substance, e.g., a sample,performing an analytical method, e.g., a method as described herein,e.g., by sample analysis of bodily fluid, such as blood by, e.g., massspectroscopy, e.g. LC-MS.

As used herein, the terms “induce” or “induction of” refer to theincrease or enhancement of a function, e.g., the increase or enhancementof the expression of a pattern recognition receptor (e.g, STING). Insome embodiments, “induction of PRR expression” refers to induction oftranscription of PRR RNA, e.g., STING RNA (e.g., mRNA, e.g., an increaseor enhancement of), or the translation of a PRR protein, e.g., the STINGprotein (e.g., an increase or enhancement of). In some embodiments,induction of PRR expression (e.g., STING expression) refers to theincrease or enhancement of the concentration of a PRR RNA, e.g., orSTING RNA (e.g., mRNA) or the STING protein, e.g., in a cell. In someembodiments, induction of PRR expression (e.g., STING expression) refersto the increase of the number of copies of PRR RNA, e.g., STING RNA(e.g., mRNA) or PRR protein, e.g., the STING protein, e.g., in a cell.In some embodiments, to induce expression of a PRR (e.g., STING) mayrefer to the initiation of PRR RNA (e.g., STING RNA (e.g., mRNA)) ortranscription or PRR protein (e.g., STING protein) translation. In someembodiments, to induce expression of a PRR (e.g., STING) may refer to anincrease in the rate of PRR RNA (e.g., STING RNA (e.g., mRNA))transcription or an increase in the rate of PRR protein (e.g., STINGprotein) expression.

As used herein, the terms “activate” or “activation” refer to thestimulation or triggering of a function, e.g., of a downstream pathway,e.g., a downstream signaling pathway. In some embodiments, activation ofa pattern recognition receptor (PRR) (e.g., STING) refers to thestimulation of a specific protein or pathway, e.g., through interactionwith a downstream signaling partner (e.g., IFN-β promoter stimulator 1(IPS-1), IRF3, IRF7, NF-κB, interferons (e.g., IFN-α or IFN-β) and/orcytokines). In some embodiments, activation is distinct from theinduction of expression of a PRR. In some embodiments, a PRR may beactivated without resulting in an induction of PRR expression (e.g.,expression of STING). In some embodiments, activation may includeinduction of expression of a PRR (e.g., STING). In some embodiments,activation of a PRR may trigger the induction of expression of a PRR(e.g., STING) by about 0.1%, about 0.5%, about 1%, about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, about 95%, or more compared to areference standard (e.g., basal expression levels of a PRR (e.g.,STING)).

As used herein, an amount of a compound, conjugate, or substanceeffective to treat a disorder (e.g., a disorder described herein),“therapeutically effective amount,” “effective amount” or “effectivecourse” refers to an amount of the compound, substance, or compositionwhich is effective, upon single or multiple dose administration(s) to asubject, in treating a subject, or in curing, alleviating, relieving orimproving a subject with a disorder (e.g., a microbial infection) beyondthat expected in the absence of such treatment.

As used herein, the terms “prevent” or “preventing” as used in thecontext of a disorder or disease, refer to administration of an agent toa subject, e.g., the administration of a compound of the presentdisclosure (e.g., compound of Formula (I)) to a subject, such that theonset of at least one symptom of the disorder or disease is delayed ascompared to what would be seen in the absence of administration of saidagent.

As used herein, the terms “reference treatment” or “reference standard”refer to a standardized level or standardized treatment that is used asbasis for comparison. In some embodiments, the reference standard orreference treatment is an accepted, well known, or well characterizedstandard or treatment in the art. In some embodiments, the referencestandard describes an outcome of a method described herein. In someembodiments, the reference standard describes a level of a marker (e.g.,a level of induction of a PRR, e.g., STING) in a subject or a sample,e.g., prior to initiation of treatment, e.g., with a compound orcomposition described herein. In some embodiments, the referencestandard describes a measure of the presence of, progression of, orseverity of a disease or the symptoms thereof, e.g., prior to initiationof treatment, e.g., with a compound or composition described herein.

As used herein, the term “subject” is intended to include human andnon-human animals. Exemplary human subjects include a human patienthaving a disorder, e.g., a disorder described herein, or a normalsubject. The term “non-human animals” includes all vertebrates, e.g.,non-mammals (such as chickens, amphibians, reptiles) and mammals, suchas non-human primates, domesticated and/or agriculturally usefulanimals, e.g., sheep, dogs, cats, cows, pigs, etc. In exemplaryembodiments of the disclosure, the subject is a woodchuck (e.g., anEastern woodchuck (Marmota monax)).

As used herein, the terms “treat” or “treating” a subject having adisorder or disease refer to subjecting the subject to a regimen, e.g.,the administration of a compound of Formula (I) or pharmaceuticallyacceptable salt thereof, or a composition comprising Formula (I) orpharmaceutically acceptable salt thereof, such that at least one symptomof the disorder or disease is cured, healed, alleviated, relieved,altered, remedied, ameliorated, or improved. Treating includesadministering an amount effective to alleviate, relieve, alter, remedy,ameliorate, improve or affect the disorder or disease, or the symptomsof the disorder or disease. The treatment may inhibit deterioration orworsening of a symptom of a disorder or disease.

As used herein, the term “Cmd” refers to the word “compound” or“Compound”, and all of the terms are used interchangeably.

Numerous ranges, e.g., ranges for the amount of a drug administered perday, are provided herein. In some embodiments, the range includes bothendpoints. In other embodiments, the range excludes one or bothendpoints. By way of example, the range can exclude the lower endpoint.Thus, in such an embodiment, a range of 250 to 400 mg/day, excluding thelower endpoint, would cover an amount greater than 250 that is less thanor equal to 400 mg/day.

Definitions

The term “alkyl,” as used herein, refers to a monovalent saturated,straight- or branched-chain hydrocarbon such as a straight or branchedgroup of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C₁-C₁₂alkyl, C₁-C₁₀ alkyl, and C₁-C₆ alkyl, respectively. Examples of alkylgroups include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl,tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, and the like.

The terms “alkenyl” and “alkynyl” are art-recognized and refer tounsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double or triple bond, respectively. Exemplary alkenyl groupsinclude, but are not limited to, —CH═CH₂ and —CH₂CH═CH₂.

The term “alkylene” refers to the diradical of an alkyl group.

The terms “alkenylene” and “alkynylene” refer to the diradicals of analkenyl and an alkynyl group, respectively.

The term “methylene unit” refers to a divalent —CH₂— group present in analkyl, alkenyl, alkynyl, alkylene, alkenylene, or alkynylene moiety.

The term “carbocyclic ring system”, as used herein, means a monocyclic,or fused, spiro-fused, and/or bridged bicyclic or polycyclic hydrocarbonring system, wherein each ring is either completely saturated orcontains one or more units of unsaturation, but where no ring isaromatic.

The term “carbocyclyl” refers to a radical of a carbocyclic ring system.Representative carbocyclyl groups include cycloalkyl groups (e.g.,cyclopentyl, cyclobutyl, cyclopentyl, cyclohexyl and the like), andcycloalkenyl groups (e.g., cyclopentenyl, cyclohexenyl,cyclopentadienyl, and the like).

The term “aromatic ring system” is art-recognized and refers to amonocyclic, bicyclic or polycyclic hydrocarbon ring system, wherein atleast one ring is aromatic.

The term “aryl” refers to a radical of an aromatic ring system.Representative aryl groups include fully aromatic ring systems, such asphenyl, naphthyl, and anthracenyl, and ring systems where an aromaticcarbon ring is fused to one or more non-aromatic carbon rings, such asindanyl, phthalimidyl, naphthimidyl, or tetrahydronaphthyl, and thelike.

The term “heteroalkyl” refers to an “alkyl” moiety wherein at least oneof the carbone molecules has been replaced with a heteroatom such as O,S, or N.

The term “heteroaromatic ring system” is art-recognized and refers tomonocyclic, bicyclic or polycyclic ring system wherein at least one ringis both aromatic and comprises a heteroatom; and wherein no other ringsare heterocyclyl (as defined below). In certain instances, a ring whichis aromatic and comprises a heteroatom contains 1, 2, 3, or 4independently selected ring heteroatoms in such ring.

The term “heteroaryl” refers to a radical of a heteroaromatic ringsystem. Representative heteroaryl groups include ring systems where (i)each ring comprises a heteroatom and is aromatic, e.g., imidazolyl,oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, thiophenyl pyrazolyl,pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl,naphthyridinyl, and pteridinyl; (ii) each ring is aromatic orcarbocyclyl, at least one aromatic ring comprises a heteroatom and atleast one other ring is a hydrocarbon ring or e.g., indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one,5,6,7,8-tetrahydroquinolinyl and 5,6,7,8-tetrahydroisoquinolinyl; and(iii) each ring is aromatic or carbocyclyl, and at least one aromaticring shares a bridgehead heteroatom with another aromatic ring, e.g.,4H-quinolizinyl. In certain embodiments, the heteroaryl is a monocyclicor bicyclic ring, wherein each of said rings contains 5 or 6 ring atomswhere 1, 2, 3, or 4 of said ring atoms are a heteroatom independentlyselected from N, O, and S.

The term “heterocyclic ring system” refers to monocyclic, or fused,spiro-fused, and/or bridged bicyclic and polycyclic ring systems whereat least one ring is saturated or partially unsaturated (but notaromatic) and comprises a heteroatom. A heterocyclic ring system can beattached to its pendant group at any heteroatom or carbon atom thatresults in a stable structure and any of the ring atoms can beoptionally substituted.

The term “heterocyclyl” refers to a radical of a heterocyclic ringsystem. Representative heterocyclyls include ring systems in which (i)every ring is non-aromatic and at least one ring comprises a heteroatom,e.g., tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl,piperidinyl, pyrrolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl,dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl,and quinuclidinyl; (ii) at least one ring is non-aromatic and comprisesa heteroatom and at least one other ring is an aromatic carbon ring,e.g., 1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl; and(iii) at least one ring is non-aromatic and comprises a heteroatom andat least one other ring is aromatic and comprises a heteroatom, e.g.,3,4-dihydro-1H-pyrano[4,3-c]pyridine, and1,2,3,4-tetrahydro-2,6-naphthyridine. In certain embodiments, theheterocyclyl is a monocyclic or bicyclic ring, wherein each of saidrings contains 3-7 ring atoms where 1, 2, 3, or 4 of said ring atoms area heteroatom independently selected from N, O, and S.

The term “saturated heterocyclyl” refers to a radical of heterocyclicring system wherein every ring is saturated, e.g., tetrahydrofuran,tetrahydro-2H-pyran, pyrrolidine, piperidine and piperazine.

“Partially unsaturated” refers to a group that includes at least onedouble or triple bond. A “partially unsaturated” ring system is furtherintended to encompass rings having multiple sites of unsaturation, butis not intended to include aromatic groups (e.g., aryl or heteroarylgroups) as herein defined. Likewise, “saturated” refers to a group thatdoes not contain a double or triple bond, i.e., contains all singlebonds.

The term “nucleobase” as used herein, is a nitrogen-containingbiological compound found linked to a sugar within a nucleoside—thebasic building blocks of deoxyribonucleic acid (DNA) and ribonucleicacid (RNA). The primary, or naturally occurring, nucleobases arecytosine (DNA and RNA), guanine (DNA and RNA), adenine (DNA and RNA),thymine (DNA) and uracil (RNA), abbreviated as C, G, A, T, and U,respectively. Because A, G, C, and T appear in the DNA, these moleculesare called DNA-bases; A, G, C, and U are called RNA-bases. Adenine andguanine belong to the double-ringed class of molecules called purines(abbreviated as R). Cytosine, thymine, and uracil are all pyrimidines.Other nucleobases that do not function as normal parts of the geneticcode are termed non-naturally occurring.

As described herein, compounds of the disclosure may contain “optionallysubstituted” moieties. In general, the term “substituted”, whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at eachposition. Combinations of substituents envisioned under this disclosureare preferably those that result in the formation of stable orchemically feasible compounds. The term “stable”, as used herein, refersto compounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

As described herein, compounds of the disclosure may contain “optionallysubstituted” moieties. In general, the term “substituted”, whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at eachposition. Combinations of substituents envisioned under this disclosureare preferably those that result in the formation of stable orchemically feasible compounds. The term “stable”, as used herein, refersto compounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Pattern Recognition Receptors

The disclosure presented herein features methods for the activation andinduction of PRR expression (e.g., STING expression) in a subject, e.g.,a subject with a proliferative disease (e.g., cancer). Patternrecognition receptors (PRRs) are a broad class of proteins whichrecognize pathogen-associated molecular patterns (PAMPs) conservedwithin pathogenic invaders. PAMPs are typically products of biosyntheticpathways that are essential to the survival and/or infectivity of thepathogen, e.g., lipopolysaccharides, glycoproteins, and nucleic acids.Recognition of PAMPs by their cognate PRRs activates signaling pathwaysthat result in the production of immune defense factors such aspro-inflammatory and anti-inflammatory cytokines, type I interferons(IFN-α, IFN-β), and/or interferon stimulated genes (ISGs). It is wellknown that induction of innate immune signaling also results in theactivation of T cell responses as well as the induction of adaptiveimmunity. These downstream immune effects are essential for clearance ofthe virus through apoptosis and killing of infected cells throughcytotoxic T lymphocytes and other defense mechanisms. It is also wellknown that interferons act on ISRE (interferon response elements) thatcan trigger the production of ISGs, which play an important role inantiviral cellular defense.

The stimulator of interferon genes (STING) is a cytosolicmicrobial-derived DNA sensor that has been shown to be particularlysensitive to double-stranded DNA and cyclic dinucleotides (e.g., cyclicdi-GMP) (Burdette, D. L. and Vance, R. E. (2013) Nat Immunol 14:19-26).Two molecules of STING form a homodimer mediated by an α-helix presentin the C-terminal dimerization domain, and molecular binding studieshave revealed that each STING dimer binds one molecule of microbialnucleic acids, e.g., DNA or a cyclic dinucleotide. Upon ligand binding,STING activates the innate immune response through interaction withRIG-I and IPS-1, resulting in interferon production (e.g., IFN-α andIFN-β) and other downstream signaling events. Since its discovery, STINGhas been shown to function as a critical sensor of viruses (e.g.,adenovirus, herpes simplex virus, hepatitis B virus, vesicularstomatitis virus, hepatitis C virus), bacteria (e.g., Listeriamonocytogenes, Legionella pneumopholia, Mycobacterium tuberculosis) andprotozoa (Plasmodium falciparum, Plasmodium berghei). In addition, STINGhas been shown to play a major role in the innate immune responseagainst tumor antigens, driving dendritic cell activation and subsequentT cell priming in several cancers (Woo, S. R. et al. Trends in Immunol(2015) 36:250-256).

Another class of PRRs includes RIG-I, which is the founding member of afamily of PRRs termed RIG-I-like receptors (RLRs) that primarily detectRNA derived from foreign sources. It is a critical sensor of microbialinfection (e.g., viral infection) in most cells and is constitutivelyexpressed at low levels in the cytosol. After ligand binding, theexpression of RIG-I is rapidly enhanced, leading to increased RIG-Iconcentrations in the cell (Jensen, S. and Thomsen, A. R. J Virol (2012)86:2900-2910; Yoneyama M. et al. Nat Immunol (2004) 5:730-737). RIG-I isan ATP-dependent helicase containing a central DExD/H box ATPase domainand tandem N-terminal caspase-recruiting domains (CARDs) that mediatedownstream signaling. The C-terminus of RIG-I comprises anssRNA/dsRNA-binding domain that when unbound acts to silence CARDfunction at the N-terminus. Without wishing to be bound by theory, it isbelieved that upon recognition of target RNA structures, two N-terminalCARDs are exposed, allowing for interaction with the CARD of adownstream binding partner, IFN-β promoter stimulator 1 (IPS-1), alsoknown as mitochondrial antiviral signaling molecule (MAVS) and CARDIF.This interaction in turn triggers further downstream signaling, such asinduction of IRF3, IRF7, NF-κB, IFNs, and cytokine production thatresults in the initiation of the host immune response.

Other RLRs are homologous to RIG-I and function in a similar manner,including MDA5, LGP2, and RNase L. MDA5 is highly homologous to RIG-I,and has been shown to be crucial for triggering a cytokine response uponinfection with picornaviruses (e.g., encephalomyocarditis virus (EMCV),Theiler's virus, and Mengo virus), Sendai virus, rabies virus, West Nilevirus, rabies virus, rotavirus, murine hepatitis virus, and murinenorovirus. LPG2 lacks a CARD domain found in RIG-I and MDA5, which isresponsible for direct interaction with IPS-1 to initiate downstreamsignaling. As such, LPG2 is believed to behave as a modulator of theinnate immune response in conjunction with other CARD-bearing RLRs suchas RIG-I and MDA5.

Another class of PRRs encompasses the nucleotide-binding andoligomerization domain (NOD)-like receptors, or NLR family (Caruso, R.et al, Immunity (2014) 41:898-908), which includes the microbial sensorNOD2. NOD2 is composed of an N-terminal CARD, a centrally-locatednucleotide-binding oligomerization domain, and a C-terminal leucine richrepeat domain that is responsible for binding microbial PAMPs, such asbacterial peptidoglycan fragments and microbial nucleic acids. Ligandbinding activates NOD2 and is believed to drive interaction with theCARD-containing kinase RIPK2, which in turn activates a number ofdownstream proteins including NF-κB, MAPK, IRF7, and IRF3, the latter ofwhich results in the induction of type 1 interferons. NOD2 is expressedin a diverse set of cell types, including macrophages, dendritic cells,paneth cells, epithelial cells (e.g., lung epithelial cells, intestinalepithelia), and osteoblasts. NOD2 has been established as a sensor ofinfection by variety of pathogenic invaders, such as protozoa (e.g.,Toxoplasma gondii and Plasmodium berghei), bacteria (e.g., Bacillusanthracis, Borrelia burgdorferi, Burkholderia pseudomallei, Helicobacterhepaticus, Legionella pneumophilia, Mycobacterium tuberculosis,Propionibacterium acne, Porphyromonas gingivalis, Salmonella enterica,and Streptococcus pneumonia), and viruses (e.g., respiratory syncytialvirus and murine norovirus-1) (Moreira, L. O. and Zamboni, D. S. FrontImmunol (2012) 3:1-12). Recent work has shown that mutation of NOD2 maycontribute to inflammatory diseases such as Crohn's disease, resultingin an aberrant inflammatory response upon stimulation.

Compounds

The present disclosure features compounds and methods for the inductionof PRR expression (e.g., STING expression) in a subject (e.g., a subjectwith a proliferative disease, e.g., a cancer), comprising administrationof a compound of Formula (I) or a prodrug or pharmaceutically acceptablesalt thereof.

In some embodiments, the present disclosure features a compound ofFormula (I) in which the 3′-OH end of one nucleoside is joined to the5′-OH of the second nucleoside through a linkage as shown. In some otherembodiments, the 2′-OH end of one nucleoside may be joined to the 5′-OHof the second nucleoside through a linkage.

In some embodiments, the compound is a compound of Formula (I):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:Z is either S or O; each of B¹ and B² is independently a purinylnucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independentlyO or S; each of Y¹ and Y² is independently O, S, or NR⁵; each of L¹ andL² is independently absent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, whereineach alkyl and heteroalkyl is optionally substituted with R⁶; each of R¹and R² is independently hydrogen, halo, —CN, C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), or OR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀alkyl (e.g., C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl),OC(O)OC₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl, heterocyclyl, aryl,or heteroaryl, wherein each alkyl, heteroalkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁸; R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl); R⁶ is halo,—CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with one ormore R⁹; ach R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), C(O)O—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)O—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), C(O)N(R⁵)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl),N(R⁵)C(O)—C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OC(O)N(R⁵)—C₁-C₂₀ alkyl(e.g., C₁-C₆ alkyl), O-aryl, O-heteroaryl, C(O)-aryl, C(O)-heteroaryl,OC(O)-aryl, C(O)O-aryl, OC(O)-heteroaryl, C(O)O-heteroaryl, C(O)O-aryl,C(O)O-heteroaryl, C(O)N(R⁵)-aryl, C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl,N(R⁵)₂C(O)-aryl, or N(R⁵)C(O)-heteroaryl, S(O)₂N(R⁵)-aryl, wherein eachalkyl, heteroalkyl, aryl, and heteroaryl is optionally substituted byone or more R⁹; and each R⁹ is independently C₁-C₂₀ alkyl, O—C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, halo, —CN, OH, oxo, aryl, heteroaryl, O-aryl,or O-heteroaryl.

In some embodiments, the compound is a compound of Formula (I-a):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of B¹ and B² is independently a purinyl nucleobase or pyrimidinylnucleobase; each of X¹ and X² is independently O or S; each of Y¹ and Y²is independently O, S, or NR⁵; each of L¹ and L² is independentlyabsent, C₁-C₂₀ alkyl or C₁-C₂₀ heteroalkyl, wherein each alkyl andheteroalkyl is optionally substituted with R⁶; each of R¹ and R² isindependently hydrogen, halo, —CN, C₁-C₂₀ alkyl, or OR⁷; each of R³ andR⁴ is independently hydrogen, C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl,cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each alkyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl isoptionally substituted with 1-5 R⁸; R⁵ is hydrogen or C₁-C₂₀ alkyl; R⁶is halo, —CN, C₁-C₂₀ alkyl, OR⁷, oxo, cycloalkyl, heterocyclyl, aryl, orheteroaryl, wherein each alkyl, cycloalkyl, heterocyclyl, aryl, orheteroaryl is optionally substituted with 1-5 R⁹; R⁷ is hydrogen, C₁-C₂₀alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein eachalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionallysubstituted with 1-5 R⁹; each R⁷ is independently C₁-C₂₀ alkyl, C₁-C₂₀heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl, C(O)O—C₁-C₂₀ alkyl,OC(O)O—C₁-C₂₀ alkyl, C(O)N(R⁵)—C₁-C₂₀ alkyl, N(R⁵)C(O)—C₁-C₂₀ alkyl,OC(O)N(R⁵)—C₁-C₂₀ alkyl, O-aryl, O-heteroaryl, C(O)-aryl,C(O)-heteroaryl, OC(O)-aryl, OC(O)-heteroaryl, C(O)O-aryl, C(O)O—heteroaryl, C(O)N(R⁵)-aryl, C(O)N(R⁵)-heteroaryl, N(R⁵)C(O)-aryl,N(R⁵)C(O)-heteroaryl, wherein each alkyl, heteroalkyl, aryl, orheteroaryl is optionally substituted by 1-5 R⁹; each R⁹ is independentlyC₁-C₂₀ alkyl, O—C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl, halo, —CN, OH, oxo,aryl, heteroaryl, O-aryl, or O-heteroaryl, wherein each alkyl,heteroalkyl, aryl.

In some embodiments, the compound is a compound of Formulas (I-b),(I-c), (I-d), or (I-e):

or a pharmaceutically acceptable salt thereof, wherein each of B¹, B²,X¹, X², Y¹, Y², L¹, L², R¹, R², R³, R⁴, and subvariables thereof aspreviously described.

In some embodiments, at least one of B¹ or B² is a purinyl nucleobase.In some embodiments, each of B¹ or B² is independently a purinylnucleobase. In some embodiments, B¹ is a purinyl nucleobase. In someembodiments, B² is a pyrimidinyl nucleobase. In some embodiments, B¹ isa purinyl nucleobase and B² is a pyrimidinyl nucleobase.

In some embodiments, each of B¹ or B² is selected from a naturallyoccurring nucleobase or a modified nucleobase. In some embodiments, eachof B¹ or B² is selected from adenosinyl, guanosinyl, cytosinyl,thyminyl, uracilyl, 5′-methylcytosinyl, 5′-fluorouracilyl,5′-propynyluracilyl, and 7-deazaadenosinyl. In some embodiments, each ofB¹ or B² is selected from:

wherein “

” indicates the linkage of the nucleobase to the ribose ring.

In some embodiments, one of B¹ or B² is selected from a naturallyoccurring nucleobase and the other of B¹ or B² is a modified nucleobase.In some embodiments, one of B¹ or B² is adenosinyl, guanosinyl,thyminyl, cytosinyl, or uracilyl, and the other of B¹ or B² is5′-methylcytosinyl, 5′-fluorouracilyl, 5′-propynyluracilyl, or7-deazaadenosinyl.

In some embodiments, B¹ is adenosinyl or guanosinyl. In someembodiments, B² is cytosinyl, thyminyl, or uracilyl. In someembodiments, B¹ is adenosinyl or guanosinyl and B² is cytosinyl,thyminyl, or uracilyl. In some embodiments, each of B¹ and B² isindependently uracilyl. In some embodiments, each of B¹ and B² isindependently adenosinyl.

In some embodiments, each of R¹ and R² is independently hydrogen, halo,or OR⁷. In some embodiments, each of R¹ and R² is independently halo(e.g., fluoro). In some embodiments, each of R¹ and R² is not hydrogenor OR⁷.

In some embodiments, X¹ is O. In some embodiments, X² is O. In someembodiments, each of X¹ and X² is independently O.

In some embodiments, Y¹ is O or S. In some embodiments, Y² is O or S. Insome embodiments, each of Y¹ and Y² is independently O or S. In someembodiments, one of Y¹ or Y² is O and the other of Y¹ or Y² is S. Insome embodiments,

each of Y¹ or Y² is independently S. In some embodiments, each of Y¹ orY² is independently O.

In some embodiments, L¹ is C₁-C₆ alkyl (e.g., CH₂). In some embodiments,L² is C₁-C₆ alkyl (e.g., CH₂). In some embodiments, each of L¹ and L² isindependently C₁-C₆ alkyl (e.g., CH₂).

In some embodiments, R³ is hydrogen, aryl, or heteroaryl, wherein aryland heteroaryl is optionally substituted with 1-5 R⁸. In someembodiments, R³ is aryl or heteroaryl, each of which is optionallysubstituted with 1-5 R⁸. In some embodiments, R³ is phenyl substitutedwith 1 R⁸.

In some embodiments, R⁴ is independently hydrogen, aryl, or heteroaryl,wherein aryl and heteroaryl is optionally substituted with 1-5 R⁸. Insome embodiments, R⁴ is aryl or heteroaryl, each of which is optionallysubstituted with 1-5 R⁸. In some embodiments, R⁴ is phenyl substitutedwith 1 R⁸.

In some embodiments, each of R³ and R⁴ is independently hydrogen, aryl,or heteroaryl, wherein aryl and heteroaryl is optionally substitutedwith 1-5 R⁸. In some embodiments, R³ is aryl or heteroaryl, each ofwhich is optionally substituted with 1-5 R⁸, and R⁴ is hydrogen. In someembodiments, R³ is phenyl substituted with 1 R⁸ and R⁴ is hydrogen. Insome embodiments, each of R³ and R⁴ is independently phenyl substitutedwith 1 R⁸.

In some embodiments, each of Y¹ and Y² is O and each of R³ and R⁴ isindependently hydrogen. In some embodiments, Y² is O and R⁴ is hydrogen.In some embodiments, each of Y¹ and Y² is independently S and each of R³and R⁴ is independently substituted with 1 R⁸. In some embodiments, Y¹is S and R³ is substituted with 1 R⁸.

In some embodiments, each R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆alkyl), C₁-C₂₀ heteroalkyl, C(O)—C₁-C₂₀ alkyl, OC(O)—C₁-C₂₀ alkyl,OC(O)O—C₁-C₂₀ alkyl, OC(O)N(R⁵)—C₁-C₂₀ alkyl, O-aryl, C(O)-aryl,OC(O)-aryl, or C(O)N(R⁵)-aryl, wherein each alkyl, heteroalkyl, aryl,and heteroaryl is optionally substituted by one or more R⁹.

In some embodiments, R⁸ is OC(O)-aryl optionally substituted by 1-5 R⁹(e.g., 1 R⁹).

In some embodiments, R⁹ is O—C₁-C₁₂ alkyl (e.g., O—CH₂(CH₂)₈CH₃). Insome embodiments, R⁹ is O—C₁-C₁₀ alkyl (e.g., O—CH₂(CH₂)₈CH₃). In someembodiments, R⁹ is O—C₁-C₈ alkyl (e.g., O—CH₂(CH₂)₆CH₃). In someembodiments, R⁹ is O—C₁-C₆ alkyl (e.g., O—CH₂(CH₂)₄CH₃).

In some embodiments, the compound is a compound of Formula (I-f):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of B¹ and B² is independently a purinyl nucleobase or pyrimidinylnucleobase; each of X¹ and X² is independently O or S; each of Y¹ and Y²is independently O, S, or NR⁵; each of L¹ and L² is independentlyabsent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, wherein each C₁-C₆ alkyl andC₁-C₆ heteroalkyl is optionally substituted with R⁶; each of R¹ and R²is independently halo; each of R³ and R⁴ is independently hydrogen,C₁-C₂₀ alkyl, C₁-C₆ heteroalkyl, cycloalkyl, heterocyclyl, aryl, orheteroaryl, wherein each C₁-C₂₀ alkyl, C₁-C₆ heteroalkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl is optionally substituted with 1-5R⁸; R⁵ is hydrogen or C₁-C₂₀ alkyl; R⁶ is halo, —CN, C₁-C₂₀ alkyl, OR⁷,oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₂₀alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionallysubstituted with 1-5 R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl, cycloalkyl,heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₂₀ alkyl,cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substitutedwith 1-5 R⁹; each R⁸ is independently C₁-C₂₀ alkyl, C(O)-aryl,C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, wherein each C₁-C₂₀alkyl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl isoptionally substituted by 1-5 R⁹; and each R⁹ is independently C₁-C₂₀alkyl, halo, —CN, OH, O—C₁-C₂₀ alkyl, O—C₁-C₂₀ heteroalkyl, O-aryl, orO-heteroaryl.

In some embodiments, the compound is a compound of Formula (I-g):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of B¹ and B² is independently a purinyl nucleobase or pyrimidinylnucleobase; each of X¹ and X² is independently O; each of Y¹ and Y² isindependently O or S; each of L¹ and L² is independently absent or C₁-C₆alkyl; each of R¹ and R² is independently halo or OH; each of R³ and R⁴is independently hydrogen or aryl optionally substituted with 1-5 R⁸;each R⁸ is independently OC(O)-aryl optionally substituted by 1-5 R⁹;and each R⁹ is independently O—C₁-C₂₀ alkyl.

In some embodiments, the compound is selected from a compound depictedin Table 1.

TABLE 1 Structure

wherein X is any pharmaceutically acceptable counterion, e.g., lithium,sodium, potassium, calcium, magnesium, aluminum, ammonium, ethylamine,diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazineand the like (see, for example, Berge et al., supra). In someembodiments, the compound of Table 1 is not a salt (e.g., is a free acidor free base).

In some embodiments, the compound is selected from a compound depictedin Table 2:

TABLE 2 Compound No. Structure Cmd 5

Cmd 16

Cmd 17

Cmd 18

Cmd 14

Cmd 12

Cmd 1

Cmd 13

Cmd 15

Cmd 4

Cmd 2

Cmd 1B

Cmd 1A

Cmd 19

Cmd 20

Cmd 21

Cmd 22

Cmd 23

Cmd 8

Cmd 3

Cmd 9

Cmd 10

Cmd 11

Cmd 24

Cmd 25

Cmd 26

Cmd 27

Cmd 28

Cmd 29

Cmd 30

Cmd 31

Cmd 32

Cmd 33

Cmd 34

Cmd 35

Cmd 36

Cmd 37

Cmd 38

Cmd 39

Cmd 40

Cmd 41

Cmd 42

Cmd 43

Cmd 44

Cmd 45

Cmd 46

Cmd 47

Cmd 48

Cmd 49

Cmd 50

Cmd 51

Cmd 52

or a pharmaceutically acceptable salt thereof.

In an embodiment, a compound described herein is in the form of apharmaceutically acceptable salt. Exemplary salts are described herein,such as ammonium salts. In some embodiments, the compound is amono-salt. In some embodiments, the compound is a di-salt. In someembodiments, a compound described herein (e.g., a compound in Table 1 orTable 2) is not a salt (e.g., is a free acid or free base).

A compound of Formula (I) or Formula (I-a) is a small molecule nucleicacid hybrid (cyclic dinucleotide) compound that combines both antiviraland immune modulating activities. The latter activity mediates, forexample, controlled apoptosis of virus-infected hepatocytes viastimulation of the innate immune response, similar to what is alsoachieved by IFN-α therapy in patients suffering from a viral infection.

Without wishing to be bound by theory, the mechanism of action of acompound of Formula (I) or Formula (I-a) entails its host immunestimulating activity, which may induce endogenous IFNs via theactivation of a PRR, e.g., RIG-I, NOD2, and STING. Activation may occurby binding of a compound of Formula (I) to the nucleotide binding domainof a PRR (e.g., STING), as described previously, and may further resultin the induction of PRR expression (e.g., STING expression).

The compounds provided herein may contain one or more asymmetric centersand thus occur as racemates and racemic mixtures, single enantiomers,individual diastereomers, and diastereomeric mixtures. All such isomericforms of these compounds are expressly included within the scope. Unlessotherwise indicated when a compound is named or depicted by a structurewithout specifying the stereochemistry and has one or more chiralcenters, it is understood to represent all possible stereoisomers of thecompound. The compounds provided herewith may also contain linkages(e.g., carbon-carbon bonds, phosphorus-oxygen bonds, orphosphorus-sulfur bonds) or substituents that can restrict bondrotation, e.g. restriction resulting from the presence of a ring ordouble bond.

In some embodiments, the method described herein comprisesadministration of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof. In some embodiments, the method describedherein comprises administration of a compound of Formula (I-a) or apharmaceutically acceptable salt thereof. In some embodiments, thecompound of Formula (I) comprises an isomer (e.g., an Rp-isomer or Spisomer) or a mixture of isomers (e.g., Rp-isomers or Sp isomers) of acompound of Formula (I). In some embodiments, the compound of Formula(I) comprises an isomer (e.g., an Rp-isomer or Sp isomer) or a mixtureof isomers (e.g., Rp-isomers or Sp isomers) of a compound of Formula(I-a).

Methods of Use

The present disclosure relates to methods for inducing the expression ofa PRR (e.g., STING) in a subject through administration of a compound ofFormula (I) or a pharmaceutically acceptable salt thereof. In someembodiments, the subject may be suffering from a condition describedbelow, e.g., a proliferative disease, e.g., a cancer.

It has been reported that many patients with advanced solid tumors showa spontaneous T cell-inflamed tumor microenvironment, which ispredictive of prognosis and clinical response to immunotherapies. Recentfindings suggest the STING pathway of cytosolic DNA sensing is animportant innate immune sensing mechanism driving type I IFN productionin the tumor context. Knowledge of this pathway is guiding the furtherdevelopment of novel immunotherapeutic strategies.

It has been reported that in early-stage colorectal cancer, the presenceof activated CD8+ T cells within the tumor microenvironment significantpositive prognostic outcome. Patients with other solid tumor histologyalso appear to have a spontaneous T cell infiltrate that may havesimilar positive prognostic value. These include breast cancer, renalcell carcinoma, melanoma, ovarian cancer, and gastrointestinal tumors.It is believed that T cell infiltrate includes tumor antigen-specific Tcells that have been activated spontaneously in response to the growingtumor, perhaps through immune surveillance mechanisms. This attemptedhost immune response, even if it does not eliminate the tumorcompletely, is thought to delay tumor progression and thus yieldimproved clinical outcome. Furthermore, the innate immune mechanisms canlead to adaptive T cell response against tumor antigens even in theabsence of exogenous infection. In this regard, human cancer geneexpression profiling studies reveal an association between a type I IFNsignature, T cell infiltration, and clinical outcome. Thus, innateimmune sensing pathways that trigger type I IFN production mightrepresent crucial intermediate mechanistic step. In gene expressionprofiling of melanoma, two major subsets of tumor microenvironment hasbeen found that represent either the presence or absence of atranscriptional profile indicative of T cell infiltrate. In fact, CD8+ Tcells, macrophages, as well as of some B cells and plasma cells in theselesions in melanoma metastases is similar to the phenotype described inearly-stage colon cancer and other tumors in which activated T cellshave been associated with favorable prognosis. CD8+ T cells wererequired for the up-regulation of all immune factors within the tumormicroenvironment. Studies indicate that IFN production is necessary foroptimal T cell priming against tumor antigens. There are many PRRs thattrigger IFN-β production by host DCs in response to a growing tumor invivo including STING. STING is an adapter protein that is activated bycyclic dinucleotides generated by cyclic GMP-AMP synthase (cGAS), whichin turn is directly activated by cytosolic DNA. In the presence of thesecyclic dinucleotides and/or DNA, STING is translocated from theendoplasmic reticulum to various perinuclear components; for example,palmitoylation of STING at the Golgi has been shown to be essential forSTING activation (Mukai, K. et al (2016) Nat Commundoi:10.1038/ncomms11932).

Activated STING forms aggregates, activates TBK1, which in turnphosphorylates interferon regulatory factor 3 (IRF3) that directlycontributes to type I IFN gene transcription. This pathway has beenimplicated in the sensing of DNA viruses, and also in selectedautoimmune models. Moreover, activating mutations of STING have recentlybeen identified in human patients with a vasculitis/pulmonaryinflammation syndrome that is characterized by increased type I IFNproduction. Mechanistic studies using mouse transplantable tumor modelsrevealed that STING-knockout mice, and IRF3-knockout mice showeddefective spontaneous T cell priming against tumor antigens in vivo, andrejection of immunogenic tumors was ablated. Similarly, tumor-derivedDNA was found within the cytosol of a major population oftumor-infiltrating DCs, and this was associated with STING pathwayactivation and IFN-β production. Therefore, the host STING pathwayappears to be an important innate immune sensing pathway that detectsthe presence of a tumor and to drive DC activation and subsequent T cellpriming against tumor-associated antigens in vivo. A functional role forthe STING pathway in vivo has also been reported in other mouse-tumorsystems. An inducible glioma model was shown to result in induction of atype I IFN gene signature as part of the host response. This inductionwas substantially reduced in STING-knockout mice, and tumors grew moreaggressively, leading to shorter mouse survival. Exogenous delivery ofcyclic dinucleotides as STING agonists exerted a therapeutic effect invivo. A crucial role for host type I IFNs and the host STING pathway wasalso confirmed in the B16. OVA and EL4. OVA models in response tocryo-ablation. Interestingly, the mechanisms involved paralleled whatwas observed in the Bm12 mouse model of lupus because host STING wasalso required for maximal production of anti-DNA antibodies. Thus, theantitumor immune response triggered in part by tumor DNA has overlapwith the mechanisms involved in autoimmunity driven by extracellularDNA. A role for STING also has been explored in an inducible coloncancer model. It seems likely that the ability of a cancer in anindividual patient to support STING pathway activation is linked to thespontaneous generation of a T cell-inflamed tumor microenvironment.Because this phenotype is associated with improved prognosis ofearly-stage cancer patients, and also with clinical response toimmunotherapies in the metastatic setting, failed STING activation maytherefore represent an early functional block, and thus itself may haveprognostic/predictive value as a biomarker. Second, strategies thatactivate or mimic the output of the host STING pathway should haveimmunotherapeutic potential in the clinic. In as much as non-Tcell-inflamed tumors appear to lack evidence of a type I IFNtranscriptional signature, strategies to promote robust innate signalingvia APCs in the tumor microenvironment might facilitate improvedcross-priming of tumor antigen-specific CD8+ T cells, and also augmentchemokine production for subsequent oncolytic activity.

Treatment of Cancer

Recognition of nucleic acid ligands by a PRRs such as cGAS, RIG-Iand/STING stimulates the production of type I interferons (e.g., IFN-αor IFN-β), thus triggering a series of downstream signaling events thatmay lead to apoptosis in susceptible cells. In recent years, aconnection between the induction of PRR expression and a number ofcancers has been discovered. For example, RIG-I expression has beenshown to be significantly downregulated in hepatocellular carcinoma, andpatients exhibiting low RIG-I expression in tumors had shorter survivaland poorer responses to IFN-α therapy (Hou, J. et al, Cancer Cell (2014)25:49-63). As such, it has been suggested that the level of RIG-Iexpression may be useful as a biomarker for prediction of prognosis andresponse to immunotherapy. In other cases, induction of RIG-I expressionhas been shown to induce immunogenic cell death of pancreatic cancercells, prostate cancer cells, breast cancer cells, skin cancer cells,and lung cancer cells (Duewell, P. et al, Cell Death Differ (2014)21:1825-1837; Besch, R. et al, J Clin Invest (2009) 119:2399-2411;Kaneda, Y. Oncoimmunology (2013) 2:e23566; Li, X. Y. et al, Mol CellOncol (2014) 1:e968016), highlighting a new approach in immune-mediatedcancer treatment.

STING is recognized as the key adapter protein in the cGAS-STING-IFNcascade, although it is also reported to be a sensor for DNA. A role forSTING in the stimulation of innate immunity in response to cancer hasalso been identified. Recent studies have revealed the presence oftumor-derived DNA in the cytosol of certain antigen-presenting cells,such as tumor-infiltrating dendritic cells, likely generated throughtumor cell stress or cell death. This tumor-derived DNA is known toactivate cGAS which causes the production of cyclic nucleotides thathave been shown to activate STING, resulting in production of associatedtype 1 interferons (Woo, S. R. et al, Immunity (2014) 41:830-842).Stimulation of STING and resulting downstream signaling pathways alsolikely contributes to effector T cell recruitment into the inflamedtumor microenvironment (Woo, S. R. Trends in Immunol (2015) 36:250-256).STING activation in the tumor microenvironment can induce adaptiveimmune response leading to anti-tumor activity. Hence, in those tumorsthat are STING-deficient, the described herein can still have anti-tumoractivity through activation of antigen-presenting cells and dendriticcells, (APCs and DCs) and induction of adaptive immune response.

In some embodiments, the methods of inducing expression of a PRR (e.g.,a PRR described herein) comprise administration of a compound of Formula(I) or a pharmaceutically acceptable salt thereof to a subject sufferingfrom cancer. In some embodiments, the methods of inducing expression ofa PRR (e.g., a PRR described herein) comprise administration of acompound of Formula (I-a) or a pharmaceutically acceptable salt thereofto a subject suffering from cancer. In some embodiments, the methods ofinducing expression of STING disclosed herein comprise administration ofa compound of Formula (I) or a pharmaceutically acceptable salt thereofto a subject suffering from cancer. In some embodiments, the methods ofinducing expression of STING disclosed herein comprise administration ofa compound of Formula (I-a) or a pharmaceutically acceptable saltthereof to a subject suffering from cancer. In some embodiments, themethods of inducing expression of RIG-I disclosed herein compriseadministration of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof to a subject suffering from cancer. In someembodiments, the methods of inducing expression of RIG-I disclosedherein comprise administration of a compound of Formula (I-a) or apharmaceutically acceptable salt thereof to a subject suffering fromcancer. In some embodiments, the methods of inducing expression of NOD2disclosed herein comprise administration of a compound of Formula (I) ora pharmaceutically acceptable salt thereof to a subject suffering fromcancer. In some embodiments, the methods of inducing expression of NOD2disclosed herein comprise administration of a compound of Formula (I-a)or a pharmaceutically acceptable salt thereof to a subject sufferingfrom cancer. In some embodiments, the cancer is selected from a cancerof the breast, bone, brain, cervix, colon, gastrointestinal tract, eye,gall bladder, lymph nodes, blood, lung, liver, skin, mouth, prostate,ovary, penis, pancreas, uterus, testicles, stomach, thymus, thyroid, orother part of the body. In some embodiments, the cancer comprises asolid tumor (e.g., a carcinoma, a sarcoma, or a lymphoma). In someembodiments, the cancer is a hepatocellular carcinoma or other cancer ofthe liver. In some embodiments, the cancer is a leukemia or other cancerof the blood. In some embodiments, the cancer comprises breast cancer,renal cell carcinoma, colon cancer, melanoma, ovarian cancer, head andneck squamous cell carcinoma, pancreatic cancer, prostate cancer, lungcancer, brain cancer, thyroid cancer, renal cancer, testis cancer,stomach cancer, urothelial cancer, skin cancer, cervical cancer,endometrial cancer, liver cancer, lung cancer, lymphoma orgastrointestinal stromal cancer and solid tumors. In some embodiments,the cancer cells (e.g., tumor cells) comprise specific cancer-associatedantigens that induce a T-cell-mediated anti-tumor response.

In some embodiments, the methods of inducing expression of a PRR (e.g.,STING, RIG-I, MDA5, LGP2) in a subject suffering from a cancer disclosedherein result in an increase in PRR expression (e.g., STING expression).In some embodiments, expression of a PRR (e.g., STING) is induced by afactor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30,about 40, about 50, about 75, about 100, about 150, about 200, about250, about 500, about 1000, about 1500, about 2500, about 5000, about10,000, or more. In some embodiments, induction of expression of a PRRse.g., STING) occurs within about 5 minutes of administration of acompound of Formula (I) or a pharmaceutically acceptable salt thereof.In some embodiments, induction of expression of a PRRs e.g., STING)occurs within about 5 minutes of administration of a compound of Formula(I-a) or a pharmaceutically acceptable salt thereof. In someembodiments, induction of expression of a PRRs (e.g., STING) occurswithin about 5 minutes of administration of a compound of Formula (I) ora pharmaceutically acceptable salt thereof. In some embodiments,induction of expression of a PRR (e.g., STING) occurs within about 10minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours,about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7hours, about 8 hours, about 10 hours, about 12 hours or more followingadministration of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof. In some embodiments, induction of expression ofa PRR (e.g., STING) occurs within about 10 minutes, about 15 minutes,about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes,about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about10 hours, about 12 hours or more following administration of a compoundof Formula (I-a) or a pharmaceutically acceptable salt thereof. It isrecognized that activation of STING by compounds may lead to inductionof expression of other PRRs such as RIG-I, MDA5, NOD2 etc. which mayfurther amplify IFN production in the tumor microenvironment and primeT-cells for enhanced anti-tumor activity.

In some embodiments, the methods of inducing expression of a PRR (e.g.,STING) in a subject suffering from a cancer disclosed herein result inan increase in PRR expression (e.g., STING expression). In someembodiments, expression of a PRR (e.g., STING) is induced by a factor ofabout 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about5, about 7.5, about 10, about 15, about 20, about 25, about 30, about40, about 50, about 75, about 100, about 150, about 200, about 250,about 500, about 1000, about 1500, about 2500, about 5000, about 10,000,or more. In some embodiments, induction of expression of a PRR (e.g.,STING) occurs within about 5 minutes of administration of a compound ofFormula (I) or a pharmaceutically acceptable salt or stereoisomerthereof. In some embodiments, induction of expression of a PRR (e.g.,STING) occurs within about 5 minutes of administration of a compound ofFormula (I-a) or a pharmaceutically acceptable salt or stereoisomerthereof. In some embodiments, induction of expression of a PRR (e.g.,STING) occurs within about 10 minutes, about 15 minutes, about 20minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours,about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10hours, about 12 hours or more following administration of a compound ofFormula (I) or a pharmaceutically acceptable salt thereof. In someembodiments, induction of expression of a PRR (e.g., STING) occurswithin about 10 minutes, about 15 minutes, about 20 minutes, about 25minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours ormore following administration of a compound of Formula (I-a) or apharmaceutically acceptable salt thereof.

Pharmaceutical Compositions

The present disclosure features methods for inducing the expression of aPRR (e.g., STING) in a subject, the methods comprising administering acompound of Formula (I), or Formula (1-a) or a pharmaceuticallyacceptable salt thereof.

While it is possible for the compound of the present disclosure (e.g., acompound of Formula (I)) to be administered alone, it is preferable toadminister said compound as a pharmaceutical composition or formulation,where the compounds are combined with one or more pharmaceuticallyacceptable diluents, excipients or carriers. The compounds according tothe disclosure may be formulated for administration in any convenientway for use in human or veterinary medicine. In certain embodiments, thecompounds included in the pharmaceutical preparation may be activeitself, or may be a prodrug, e.g., capable of being converted to anactive compound in a physiological setting. Regardless of the route ofadministration selected, the compounds of the present disclosure, whichmay be used in a suitable hydrated form, and/or the pharmaceuticalcompositions of the present disclosure, are formulated into apharmaceutically acceptable dosage form such as described below or byother conventional methods known to those of skill in the art.

The amount and concentration of compounds of the present disclosure(e.g., a compound of Formula (I)) in the pharmaceutical compositions, aswell as the quantity of the pharmaceutical composition administered to asubject, can be selected based on clinically relevant factors, such asmedically relevant characteristics of the subject (e.g., age, weight,gender, other medical conditions, and the like), the solubility ofcompounds in the pharmaceutical compositions, the potency and activityof the compounds, and the manner of administration of the pharmaceuticalcompositions. For further information on Routes of Administration andDosage Regimes the reader is referred to Chapter 25.3 in Volume 5 ofComprehensive Medicinal Chemistry (Corwin Hansch; Chairman of EditorialBoard), Pergamon Press 1990.

Thus, another aspect of the present disclosure provides pharmaceuticallyacceptable compositions comprising a therapeutically effective amount orprophylactically effective amount of a compound described herein (e.g.,a compound of Formula (I)), formulated together with one or morepharmaceutically acceptable carriers (additives) and/or diluents. Asdescribed in detail below, the pharmaceutical compositions of thepresent disclosure may be specially formulated for administration insolid or liquid form, including those adapted for oral, intratumoral,parenteral administration, for example, by subcutaneous, intramuscular,intraperitoneal, or intravenous injection as, for example, a sterilesolution or suspension. However, in certain embodiments the subjectcompounds may be simply dissolved or suspended in sterile water. Incertain embodiments, the pharmaceutical preparation is non-pyrogenic,i.e., does not elevate the body temperature of a patient.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of the compound other than directly intothe central nervous system, such that it enters the patient's systemand, thus, is subject to metabolism and other like processes, forexample, subcutaneous administration.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, stabilizing agent, excipient, solventor encapsulating material, involved in carrying or transporting thesubject antagonists from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically acceptable carriers include, but are notlimited to: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) ascorbic acid; (17)pyrogen-free water; (18) isotonic saline; (19) Ringer's solution; (20)ethyl alcohol; (21) phosphate buffer solutions; (22) cyclodextrins suchas Captisol®; and (23) other non-toxic compatible substances such asantioxidants and antimicrobial agents employed in pharmaceuticalformulations.

As set out above, certain embodiments of the compounds described hereinmay contain a basic functional group, such as an amine, and are thuscapable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present disclosure.These salts can be prepared in situ during the final isolation andpurification of the compounds of the disclosure, or by separatelyreacting a purified compound of the disclosure in its free base formwith a suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like(see, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19).

In other cases, the compounds of the present disclosure may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of the compound of the present disclosure (e.g., a compound ofFormula (I). These salts can likewise be prepared in situ during thefinal isolation and purification of the compounds, or by separatelyreacting the purified compound in its free acid form with a suitablebase, such as the hydroxide, carbonate or bicarbonate of apharmaceutically acceptable metal cation, with ammonia, or with apharmaceutically acceptable organic primary, secondary or tertiaryamine. Representative alkali or alkaline earth salts include thelithium, sodium, potassium, calcium, magnesium, and aluminum salts andthe like. Representative organic amines useful for the formation of baseaddition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like (see, for example,Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

The pharmaceutically acceptable carriers, as well as wetting agents,emulsifiers, lubricants, coloring agents, release agents, coatingagents, sweetening, flavoring agents, perfuming agents, preservatives,antioxidants, and other additional components may be present in anamount between about 0.001% and 99% of the composition described herein.For example, said pharmaceutically acceptable carriers, as well aswetting agents, emulsifiers, lubricants, coloring agents, releaseagents, coating agents, sweetening, flavoring agents, perfuming agents,preservatives, antioxidants, and other additional components may bepresent from about 0.005%, about 0.01%, about 0.05%, about 0.1%, about0.25%, about 0.5%, about 0.75%, about 1%, about 1.5%, about 2%, about3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 85%, about 90%, about 95%, or about 99% of the compositiondescribed herein.

Pharmaceutical compositions of the present disclosure may be in a formsuitable for oral administration, e.g., a liquid or solid oral dosageform. In some embodiments, the liquid dosage form comprises asuspension, a solution, a linctus, an emulsion, a drink, an elixir, or asyrup. In some embodiments, the solid dosage form comprises a capsule,tablet, powder, dragée, or powder. The pharmaceutical composition may bein unit dosage forms suitable for single administration of precisedosages. Pharmaceutical compositions may comprise, in addition to thecompound described herein (e.g., a compound of Formula (I)) or apharmaceutically acceptable salt thereof, a pharmaceutically acceptablecarrier, and may optionally further comprise one or morepharmaceutically acceptable excipients, such as, for example,stabilizers (e.g., a binder, e.g., polymer, e.g., a precipitationinhibitor, diluents, binders, and lubricants.

In some embodiments, the composition described herein comprises a liquiddosage form for oral administration, e.g., a solution or suspension. Inother embodiments, the composition described herein comprises a soliddosage form for oral administration capable of being directly compressedinto a tablet. In addition, said tablet may include other medicinal orpharmaceutical agents, carriers, and or adjuvants. Exemplarypharmaceutical compositions include compressed tablets (e.g., directlycompressed tablets), e.g., comprising a compound of the presentdisclosure (e.g., a compound of Formula (I)) or a pharmaceuticallyacceptable salt thereof.

Formulations of the present disclosure include those suitable forparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about1 percent to about 99 percent of active ingredient, preferably fromabout 5 percent to about 70 percent, most preferably from about 10percent to about 30 percent. Pharmaceutical compositions of thisdisclosure suitable for parenteral administration comprise compounds ofthe disclosure in combination with one or more pharmaceuticallyacceptable sterile isotonic aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, or sterile powders which may bereconstituted into sterile injectable solutions or dispersions justprior to use, which may contain antioxidants, buffers, bacteriostats,solutes which render the formulation isotonic with the blood of theintended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a compound of thepresent disclosure (e.g., a compound of Formula (I)), it may bedesirable to slow the absorption of the drug from subcutaneous,intraperitoneal, or intramuscular injection. This may be accomplished bythe use of a liquid suspension of crystalline or amorphous materialhaving poor water solubility. The rate of absorption of the drug thendepends upon its rate of dissolution, which, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered form of the compound of the presentdisclosure is accomplished by dissolving or suspending compound in anoil vehicle.

In some embodiments, it may be advantageous to administer the compoundof the present disclosure (e.g., a compound of Formula (I)) in asustained fashion. It will be appreciated that any formulation thatprovides a sustained absorption profile may be used. In certainembodiments, sustained absorption may be achieved by combining acompound of the present disclosure with other pharmaceuticallyacceptable ingredients, diluents, or carriers that slow its releaseproperties into systemic circulation.

Routes of Administration

The compounds and compositions used in the methods described herein maybe administered to a subject in a variety of forms depending on theselected route of administration, as will be understood by those skilledin the art. Exemplary routes of administration of the compositions usedin the methods described herein include topical, enteral, or parenteralapplications. Topical applications include but are not limited toepicutaneous, inhalation, enema, eye drops, ear drops, and applicationsthrough mucous membranes in the body. Enteral applications include oraladministration, rectal administration, vaginal administration, andgastric feeding tubes. Parenteral administration includes intravenous,intraarterial, intracapsular, intraorbital, intracardiac, intradermal,transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural, intrasternal, intraperitoneal, subcutaneous,intramuscular, transepithelial, nasal, intrapulmonary, intrathecal,rectal, and topical modes of administration. Parenteral administrationmay be by continuous infusion over a selected period of time. In certainembodiments of the disclosure, a composition described herein comprisinga compound of Formula (I) is administered orally. In certain embodimentsof the disclosure, a composition described herein comprising a compoundof Formula (I-a) is administered orally. In other embodiments of thedisclosure, a composition described herein comprising a compound ofFormula (I) is administered parenterally (e.g., intraperitoneally). Itis recognized that for treatment of solid tumors, direct injection ofthe compounds into the tumor may also be carried out (e.g., intratumoraladministration). In other embodiments of the disclosure, a compositiondescribed herein comprising a compound of Formula (I-a) is administeredparenterally (e.g., intraperitoneally). It is recognized that fortreatment of solid tumors, direct injection of the compounds into thetumor may also be carried out (e.g., intratumoral administration).

For intravenous, intraperitoneal, or intrathecal delivery or directinjection (e.g., intratumoral), the composition must be sterile andfluid to the extent that the composition is deliverable by syringe. Inaddition to water, the carrier can be an isotonic buffered salinesolution, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. Proper fluidity can be maintained, for example, by use ofcoating such as lecithin, by maintenance of required particle size inthe case of dispersion and by use of surfactants. In many cases, it ispreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol or sorbitol, and sodium chloride in the composition.Long-term absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

The choice of the route of administration will depend on whether a localor systemic effect is to be achieved. For example, for local effects,the composition can be formulated for topical administration and applieddirectly where its action is desired. For systemic, long term effects,the composition can be formulated for enteral administration and givenvia the digestive tract. For systemic, immediate and/or short termeffects, the composition can be formulated for parenteral administrationand given by routes other than through the digestive tract.

Dosages

The compositions of the present disclosure are formulated intoacceptable dosage forms by conventional methods known to those of skillin the art. Actual dosage levels of the active ingredients in thecompositions of the present disclosure (e.g., a compound of Formula (I))may be varied so as to obtain an amount of the active ingredient whichis effective to achieve the desired therapeutic response for aparticular subject, composition, and mode of administration, withoutbeing toxic to the subject. The selected dosage level will depend upon avariety of pharmacokinetic factors including the activity of theparticular compositions of the present disclosure employed, the route ofadministration, the time of administration, the rate of absorption ofthe particular agent being employed, the duration of the treatment,other drugs, substances, and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the subject being treated,and like factors well known in the medical arts. A physician orveterinarian having ordinary skill in the art can readily determine andprescribe the effective amount of the composition required. For example,the physician or veterinarian can start doses of the substances of thedisclosure employed in the composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved. Ingeneral, a suitable daily dose of a composition of the disclosure willbe that amount of the substance which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Preferably, the effective dailydose of a therapeutic composition may be administered as two, three,four, five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms.

Preferred therapeutic dosage levels are between about 0.1 mg/kg to about1000 mg/kg (e.g., about 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg,200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg) of thecomposition per day administered (e.g., orally or intraperitoneally) toa subject afflicted with the disorders described herein (e.g., HBVinfection). Preferred prophylactic dosage levels are between about 0.1mg/kg to about 1000 mg/kg (e.g., about 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg,1.5 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000mg/kg) of the composition per day administered (e.g., orally orintraperitoneally) to a subject. The dose may also be titrated (e.g.,the dose may be escalated gradually until signs of toxicity appear, suchas headache, diarrhea, or nausea).

The frequency of treatment may also vary. The subject can be treated oneor more times per day (e.g., once, twice, three, four or more times) orevery so-many hours (e.g., about every 2, 4, 6, 8, 12, or 24 hours). Thecomposition can be administered 1 or 2 times per 24 hours. The timecourse of treatment may be of varying duration, e.g., for two, three,four, five, six, seven, eight, nine, ten, or more days, two weeks, 1month, 2 months, 4 months, 6 months, 8 months, 10 months, or more thanone year. For example, the treatment can be twice a day for three days,twice a day for seven days, twice a day for ten days. Treatment cyclescan be repeated at intervals, for example weekly, bimonthly or monthly,which are separated by periods in which no treatment is given. Thetreatment can be a single treatment or can last as long as the life spanof the subject (e.g., many years).

Patient Selection and Monitoring

The methods of the present disclosure described herein entailadministration of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof to a subject to activate the PRR for IFNs, ISGsand cytokines production or additionally induce the expression of PRRs(e.g., RIG-I, STING etc.). In some embodiments, the subject is sufferingfrom or is diagnosed with a condition, e.g., a proliferative disease,e.g., cancer. Accordingly, a patient and/or subject can be selected fortreatment using a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof by first evaluating the patient and/or subjectto determine whether the subject is infected with a proliferativedisease, e.g., cancer. A subject can be evaluated as infected with aproliferative disease (e.g., cancer) using methods known in the art. Thesubject can also be monitored, for example, subsequent to administrationof a compound described herein (e.g., a compound of Formula (I) or apharmaceutically acceptable salt thereof.

In some embodiments, the subject is a mammal. In some embodiments, thesubject is a human. In some embodiments, the subject is an adult. Insome embodiments, the subject has a proliferative disease, e.g., cancer.In some embodiments, the subject has a cancer of the of the breast,bone, brain, cervix, colon, gastrointestinal tract, eye, gall bladder,lymph nodes, blood, lung, liver, skin, mouth, prostate, ovary, penis,pancreas, uterus, testicles, stomach, thymus, thyroid, or other part ofthe body. In some embodiments, the subject has a cancer comprising asolid tumor (e.g., a carcinoma, a sarcoma, or a lymphoma). In someembodiments, the subject has a hepatocellular carcinoma or other cancerof the liver. In some embodiments, the subject has a leukemia or othercancer of the blood. In some embodiments, the subject has a breastcancer, renal cell carcinoma, colon cancer, melanoma, ovarian cancer,head and neck squamous cell carcinoma, pancreatic cancer, prostatecancer, lung cancer, brain cancer, or gastrointestinal stromal cancer.In some embodiments, the subject has cancer cells (e.g., tumor cells)comprising specific cancer-associated antigens that induce a T-cellresponse.

In some embodiments, the subject is treatment naïve. In someembodiments, the subject has been previously treated for a proliferativedisease (e.g., a cancer). In some embodiments, the subject has relapsed.

Combination Therapies

A compound described herein may be used in combination with other knowntherapies. Administered “in combination”, as used herein, means that two(or more) different treatments are delivered to the subject during thecourse of the subject's affliction with the disorder, e.g., the two ormore treatments are delivered after the subject has been diagnosed withthe disorder and before the disorder has been cured or eliminated ortreatment has ceased for other reasons. In some embodiments, thedelivery of one treatment is still occurring when the delivery of thesecond begins, so that there is overlap in terms of administration. Thisis sometimes referred to herein as “simultaneous” or “concurrentdelivery”. In other embodiments, the delivery of one treatment endsbefore the delivery of the other treatment begins. In some embodimentsof either case, the treatment is more effective because of combinedadministration. For example, the second treatment is more effective,e.g., an equivalent effect is seen with less of the second treatment, orthe second treatment reduces symptoms to a greater extent, than would beseen if the second treatment were administered in the absence of thefirst treatment, or the analogous situation is seen with the firsttreatment. In some embodiments, delivery is such that the reduction in asymptom, or other parameter related to the disorder is greater than whatwould be observed with one treatment delivered in the absence of theother. The effect of the two treatments can be partially additive,wholly additive, or greater than additive. The delivery can be such thatan effect of the first treatment delivered is still detectable when thesecond is delivered.

A compound described herein and the at least one additional therapeuticagent can be administered simultaneously, in the same or in separatecompositions, or sequentially. For sequential administration, thecompound described herein can be administered first, and the additionalagent can be administered second, or the order of administration can bereversed.

In some embodiments, the combination of a compound of Formula (I) or apharmaceutically acceptable salt thereof and the additional agent has asynergistic or additive effect. In some embodiments, the term “additive”refers to an outcome wherein when two agents are used in combination,the combination of the agents acts in a manner equal to but not greaterthan the sum of the individual activity of each agent.

In some embodiments, the combination of a compound of Formula (I-a) or apharmaceutically acceptable salt thereof and the additional agent has asynergistic or additive effect. In some embodiments, the term “additive”refers to an outcome wherein when two agents are used in combination,the combination of the agents acts in a manner equal to but not greaterthan the sum of the individual activity of each agent. In someembodiments, the terms “synergy” or “synergistic” refer to an outcomewherein when two agents are used in combination, the combination of theagents acts so as to require a lower concentration of each individualagent than the concentration required to be efficacious in the absenceof the other agent. In some embodiments, a synergistic effect results ina reduced in a reduced minimum inhibitory concentration of one or bothagents, such that the effect is greater than the sum of the effects. Asynergistic effect is greater than an additive effect. In someembodiments, the agents in the composition herein may exhibit asynergistic effect, wherein the activity at a particular concentrationis greater than at least about 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 10, 12,15, 20, 25, 50, or 100 times the activity of either agent alone.

For example, any of the methods described herein may further comprisethe administration of a therapeutically effective amount of anadditional agent. Exemplary additional pharmaceutical agents include,but are not limited to, anti-proliferative agents, anti-cancer agents,anti-diabetic agents, anti-inflammatory agents, immunosuppressantagents, and a pain-relieving agent. Pharmaceutical agents include smallorganic molecules such as drug compounds (e.g., compounds approved bythe U.S. Food and Drug Administration as provided in the Code of FederalRegulations (CFR)), peptides, proteins, carbohydrates, monosaccharides,oligosaccharides, polysaccharides, nucleoproteins, mucoproteins,lipoproteins, synthetic polypeptides or proteins, small molecules linkedto proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs,nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides,lipids, hormones, vitamins, and cells. In some embodiments, theadditional agent is an anti-cancer agent, e.g., an alkylating agent(e.g., cyclophosphamide).

In an embodiment, the additional agent is an immunooncology agent, forexample, an agent that activate the immune system, e.g., making it ableto recognize cancer cells and destroy them. Exemplary immonooncologycompounds are compounds that inhibit the immune checkpoint blockadepathway. In an embodiment, the compound is an antibody such as a PD-1 orPD-L1 antibody or a co-stimulatory antibody. In some embodiments, thecompound is an anti-CTLA4 antibody. In another embodiment, the agent isa cell based agent such as CAR-t therapy.

EXAMPLES

The disclosure is further illustrated by the following examples andsynthesis schemes, which are not to be construed as limiting thisdisclosure in scope or spirit to the specific procedures hereindescribed. It is to be understood that the examples are provided toillustrate certain embodiments and that no limitation to the scope ofthe disclosure is intended thereby. It is to be further understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which may suggest themselves to those skilled in theart without departing from the spirit of the present disclosure and/orscope of the appended claims.

Abbreviations used in the following examples and elsewhere herein are:

-   -   DCA dichloroacetic acid    -   DCC N,N′-dicyclohexylcarbodiimide    -   DCM dichloromethane    -   DMAP 4-dimethylaminopyridine    -   ETT 5-(ethylthio)-1H-tetrazole    -   h hours    -   IPA isopropyl alcohol    -   LCMS liquid chromatography-mass spectrometry    -   MeOH methanol    -   PTSA p-Toluenesulfonic acid    -   r.t. room temperature    -   THE tetrahydrofuran    -   TLC thin-layer chromatography

Example 1. Synthesis of Exemplary Compounds of the Disclosure

Procedure for Synthesis of Cyclic Dinucleotide Prodrug 9 and 4 andCyclic Thio-Diphosphates

Synthesis of 5′-OH-3′-Levulinyl-2′F-dA: Levulinic acid (2.148 g, 18.5mmol) was dissolved in dry-dioxane (50 mL) and the solution was cooledto 5-10° C. on an ice-water bath. DCC (1.939 g, 9.4 mmol) was addedportion wise over 1 h. The ice-water bath was removed and the reactionwas allowed to warm to room temperature over 2 hours. The resultingdicyclohexyl urea precipitate was filtered off, and washed withdry-dioxane (10 mL). The filtrate was added to a solution of5′DMT-2′F-3′OH-dA (5.0 g, 7.4 mmol) in dry pyridine (50 mL) and acatalytic amount of DMAP then was added under atmosphere of argon. Afterstirring for 2 hours at room temperature, the mixture was evaporated todryness. The residue was dissolved in DCM (150 mL) and the organic phasewas washed with 5% NaHCO₃ (100 mL) and brine (100 mL), dried over Na₂SO₄and concentrated under reduced pressure to provide the desired productas a white solid.

Detritylation: Above solid was dissolved in DCM (100 mL), and water(1.33 mL, 74 mmol) was added to reaction mixture. 6% DCA in DCM (100 mL)was then added and the reaction mixture was stirred at room temperaturefor 10-15 min. The resulting mixture was quenched by the addition ofmethanol (25 mL) and then washed with 5% NaHCO₃ solution (150 mL) andbrine (150 mL). The combined organic layers were dried over Na₂SO₄ andconcentrated under reduced pressure. The crude residue was purifiedusing combi-flash silicagel column chromatography eluting with 0-5% MeOHin DCM to give 3.45 g (62% yield) of pure desired product as a whitesolid.

Coupling: 5′OH-3′-Levulinylated-2′F-deoxy-Adinosine (700 mg, 1.48 mmol)and 5′DMT-2′F-3′CED-Phosphoamidite-deoxy-Uridine (1.66 g, 2.22 mmol)mixture was dried under high vacuum for 1-2 hours. Argon was flushedover the round bottom flask containing reaction mixture. Anhydrousacetonitrile (40 mL) was added to reaction mixture Followed by ETT (279mg, 2.146 mmol) in acetonitrile (5.0 mL) under atmosphere of argon. Theresulting mixture was stirred at room temperature under argon for 2 h.Once TLC analysis showed reaction completion, water was added (80 μL, 2equivalents to amidite).

Sulfurization: In a silanized flask, Beaucage reagent (3H-BD) (592 mg,2.96 mmol) was dissolved in acetonitrile (5.0 mL). The above couplingreaction mixture was transferred to solution of sulfurizing reagent(3H-BD) in acetonitrile and under an atmosphere of argon. The resultingmixture was stirred at room temperature for 45 min. to complete thesulfurization reaction. Methanol (10 mL) was added and the reactionmixture was then stirred for 30 min. The resulting mixture wasevaporated under reduced pressure to dryness. The crude residue wasdissolved in DCM (100 mL) and washed with water (75 mL). DCM layer wasseparated, dried over Na₂SO₄ and used for in the detritylation step.

Detritylation: The above obtained DCM layer containing the sulfurizationproduct was cooled in an ice-water bath. 5% PTSA solution in DCM:MeOH(7:3, 100 mL) was added and the reaction mixture was stirred for 15 min.to complete the detritylation reaction. Water (50 mL) was then added andthe resulting mixture was stirred for another 15 minutes. The reactionmixture was transferred to separator funnel and the water was layer wasseparated. The organic layer was washed 5% NaHCO₃ solution (100 mL), pHof the aqueous layer is above 7.0. The combined organic layers weredried over Na₂SO₄ and concentrated under reduced pressure to give thecrude product. The crude product was purified using combiflash silicagelcolumn chromatography eluting with 0-5% MeOH in DCM to give 960 mg ofpure desired product as a white solid.

Levulinyl group deprotection: 3′-Levulinyl protected dinucleotidethiophosphate was treated with 0.5M hydrazine monohydrate in a mixtureof pyridine: acetic acid (3:2 and the reaction mixture stirred at roomtemperature for 15 minutes. Once TLC analysis showed reactioncompletion, 2,4-pentanedione (2.0, mL) was then added to quenchunreacted hydrazine hydrate. The volatiles were removed under reducedpressure and the reaction mixture was partitioned between 25% IPA in DCM(50 mL) and water (50 mL). The organic layers were collected andevaporated to dryness under reduced pressure to give thick liquid, whichwas co-evaporated with toluene (2×15 mL) to provide crude residue whichwas purified on Combiflash silicagel column chromatography using 0-10%MeOH in DCM to give 725 mg of pure desired product as a white solid.

Cyclization: Dinucleotide phosphorothioate trimester (1 equivalent) and2-cyanoethyl tetra isopropyl phosphorodiamidite (bisamidite) (1equivalent) were dissolved in a mixture of dry acetonitrile and dry DCM(2:1, 30 mL). Disopropylaminotetrazolide (1 equivalent) was added toreaction mixture in 4 portions over a period of 1 hour under an inertatmosphere. The solution was stirred for an additional 2 h at r.t. andETT (2.0 equivalent) was then added to the reaction mixture was stirredfor overnight. Deoxygenated water (29 μL) was then added to reactionmixture.

Sulfurization (Synthesis of protected cyclic phosphorothiodiphosphate):Beaucage reagent (3H-BD) (2.0 equivalent) was dissolved in acetonitrilein a silanized flask. One portion of above cyclization product (twothirds) was added to sulfurizing reagent under an atmosphere of argon.and the reaction mixture was stirred at room temperature for 45 minutes.Methanol (10 mL) was then added and the resulting mixture was stirredfor 30 minutes. Solvents were evaporated under reduced pressure and thecrude residue was dissolved in DCM (50 mL) and washed with water (50mL). The DCM layers were separated, dried over Na₂SO₄ and concentratedunder reduced pressure. The crude product was purified using Combiflashsilica gel column chromatography eluting with 0-10% MeOH in DCM to give150 mg of pure desired product.

Oxidation (Synthesis of protected cyclic phosphoromonothio diphosphate):TBHP (4.0 equivalent) was added to a stirred solution of a secondportion of cyclization product (one third) at 0° C. and reaction mixturewas warmed to r.t. over 15 minutes. Excess TBHP was quenched by additionof a saturated sodium bisulfite solution and the resulting mixture wasevaporated under reduced pressure. The crude residue was dissolved inDCM (25 mL) and washed with water (20 mL). Organic layers were separatedand dried over Na₂SO₄ and concentrated under reduced pressure. Theresulting crude product was purified using Combiflash silicagel columnchromatography eluting with 0-10% MeOH in DCM to give 60 mg of puredesired product.

Deprotection of cyclic phosphorothiodiphosphate [Synthesis of Cmd 2]:Protected cyclic phosphorothiodiphosphate (60 mg) was dissolved in conc.NH₄OH (2.0 mL) and stirred at r.t. overnight. Once LCMS showed reactioncompletion, the mixture was evaporated under reduced pressure to removeammonia. The water layer was washed with ethyl acetate (5×5 mL),separated and lyophilized to provide 100 mg of crude product as a whitefluffy solid.

Alkylation of cyclic phosphorothio diphosphate [Synthesis of Cmd 4]:Cyclic phosphorothio diphosphate (25 mg) was dissolved in water (250μL). A solution of 4-(iodomethyl)phenyl 4-(decyloxy)benzoate (42 mg) ina mixture of THF:Acetone (1:1, 2.0 mL) was then added. Reaction mixturepH was approximately 3.5-4.0. The reaction mixture was stirred at r.t.for 40 hours. The crude product was purified using Combiflash silicagelcolumn chromatography eluting 0-10% IPA in DCM to give 25 mg of thedesired product as a yellowish brown solid.

Deprotection of cyclic phosphoromonothio diphosphate [Synthesis of Cmd3]: Protected cyclic phosphoro monothio diphosphate (60 mg) wasdissolved in conc. NH₄OH (5.0 mL) and then stirred at r.t. forovernight. Once LCMS showed reaction, the mixture was evaporated underreduced pressure to remove ammonia. The water layer was washed withethyl acetate (5×5 mL), separated and lyophilized to provide 50 mg ofthe crude desired product as a white fluffy solid.

Alkylation of cyclic phosphoromonothio diphosphate [Synthesis of Cmd 1]:Cyclic phosphoromonothio diphosphate (20 mg) was dissolved in water (200μL). A solution of 4-(iodomethyl)phenyl 4-(decyloxy)benzoate (18 mg) ina mixture of THF:Acetone (1:1, 1.4 mL) was then added. The reactionmixture pH was approximately 4.0. The reaction mixture stirred at r.t.overnight and solvents were removed under reduced pressure. Theresulting crude residue was redissolved in water:acetonitrile (1:1, 2.0mL). A precipitate (unreacted alkylating reagent) formed and was removedby centrifugation. The mother liquor was lyophilized and the crudeproduct was purified by using C₁₈ sep pack column (Waters, 4.0 g) with0.2M ammonium acetate buffer. The compound was eluted withacetonitrile:water (1:1). The pure fractions were collected andlyophilized to provide 5-6 mg of pure desired product as a white fluffysolid.

Example 2. In Vitro Activation of ISG54 and NF-Kp in H1EK293 Cells

In this experiment, HTEK293 cells (SZ14) stably expressing either theISG54 ISRE-luc reporter or the NF-κβ-luc reporter gene were treated induplicate with an exemplary compound of the disclosure or 2′,3′-cGAMP asa control, each in digitonin buffer for 5 hours, in order to screen forpotential STING agonists. ISG54 or NF-κβ activity was determined usingthe Steady-glo buffer system (Promega), and are expressed as EC₅₀ valuessummarized in Table 3 below. In general, half maximal effectiveconcentration (EC₅₀) refers to the concentration of a drug that inducesa response halfway between the baseline and maximum after a specifiedexposure time. This calculation is applicable for compounds with enzymeinhibition activity, as the baseline for an untreated sample may be setat 100% o enzymatic activity, and therefore % inhibition is evaluatedbased on this 100% maximal basis. For these studies, the EC₅₀ valuerelates to the concentration required to achieve a value 50% o activitylevel above the untreated sample set at 0%.

In Table 3, “A” represents an EC₅₀ of less than 50 nM; “B” an EC₅₀ ofbetween 50 nM and 500 nM; “C” an EC₅₀ of between 500 nM and 1 μM; “D” anEC₅₀ of between 1 μM and 2 μM; and “E” an EC₅₀ of greater than 2 μM.Data are shown as fold induction over cells that received DMSO (compoundcardier) alone as the mean, +/−standard deviation of duplicate wells perstimulant.

TABLE 3 EC₅₀ values for exemplary compounds of the disclosure CompoundNo. IRF EC₅₀ NF-KB EC₅₀ Cmd 5 E E Cmd 17 E E Cmd 18 E E Cmd 14 A A Cmd12 A A Cmd 1 A B Cmd 13 A B Cmd 15 A A Cmd 4 D E Cmd 2 D E Cmd 1B A BCmd 1A A B Cmd 20 E E Cmd 21 C E Cmd 22 A B Cmd 23 A B Cmd 25 A B Cmd 26A A Cmd 29 B C Cmd 30 C E Cmd 31 C E Cmd 32 B B

Example 3. Evaluation of IRF-Type I IFN Activity in THP Cells

THP1-dual cells were treated in triplicate with exemplary compounds ofthe disclosure in lipofectamine (e.g., compound 2 or compound 3) or2′,3′-cGAMP in lipofectamine as a control at varying concentrations for22 hours. Levels of IRF-inducible luciferase reporter activity in thecell culture supernatants were assayed using the Quanti-luc reagent, andare summarized in FIG. 9 . Data are shown as fold induction over cellsthat received DMSO (compound carrier) alone as the mean, +/−standarddeviation of duplicate wells per stimulant.

Example 4. Determination of Cytotoxicity of Exemplary Compounds

The cytotoxicity of exemplary compounds in THP1 cells was assessed usingCell titer Glo Assay (Promega). THP1 dual cells grown in complete mediawere treated with various concentrations of compounds or DMSO control.The CellTiter-Glo® Luminescent Cell Viability/cytotoxicity was adetermined by assessing number of viable cells in culture based onquantitation of the ATP present through a “glow-type” luminescentsignal, produced by the luciferase reaction. % apoptosis was calculatedfrom fold change in luminescence compared to DMSO treated sample.

Example 5. Quantification of STING Binding

SZ14 HEK293 cells stably expressing the ISG54 ISRE-luc reporter genewere treated with compound exemplary compounds Cmd 1, 2′3′-cGAMP(natural STING ligand), or DMSO in the presence of digitonin for 5-6hrs. ISRE-luciferase activity was determined and normalized to DMSOtreated cells (mean±standard deviation of triplicate wells perstimulant).

Alternatively, raw-ISG-Dual cells in 96-well plates were stimulated intriplicate with compound/lipo, cGAMP/lipo complex or compound alone for22-24 hrs at 37° C., 5% CO2. Activity of secreted luciferase in cellculture supernatant was measured using Invivogen Quanti-luc. Data areshown as fold induction over DMSO treated cells (mean±standard deviationof triplicate wells per stimulant).

Example 6. Induction of Type III IFN (IL-29) Production in THP Cells byExemplary Compounds

THP1-Dual (WT) cells were treated in triplicate with an exemplarycompound alone or cGAMP/lipo for 21 hrs. Level of IL-29 in culturesupernatant was determined using ELISA. Results shown are theaverage±standard deviation of duplicate wells.

Example 7. FIG. 9 Shows that Cmd 1 Causes Cell Death by Apoptosis

The apoptosis in THP1 cells was assessed using Caspase-Glo® 3/7 Assay(Promega). THP1 dual cells grown in complete media were treated withvarious concentrations of Cmd 1 or 2′3-cGAMP or DMSO control withLipofectamine LTX. The caspase-3 and -7 activity was measured by using apro-luminescent caspase-3/7 substrate that contains the tetrapeptidesequence DEVD which is cleaved to release amino-luciferin, a substrateof luciferase used in the production of light. After incubation for 20h, Apoptotic activity was assessed by measuring levels ofamino-luciferin. % Apoptosis was calculated from fold-change inluminescence compared to DMSO-treated sample. CC50 values are generatedby curve fit in Xlfit.

Example 8. FIG. 10 Shows the Selective Induction of Apoptosis by Cmd 1in Acute Monocytic Leukemia Cell Line (THP1) Vs. PBMCs

The Apoptosis in THP1 cells and PBMCs was assessed using Caspase-Glo®3/7 Assay (Promega). THP1 cells and PBMCs grown in complete media weretreated with various concentrations of Cmd 1 or 2′3-cGAMP or DMSOcontrol with Lipofectamine LTX. The caspase-3 and -7 activity wasmeasured by using a proluminescent caspase-3/7 substrate that containsthe tetrapeptide sequence DEVD which will be cleaved to releaseaminoluciferin, a substrate of luciferase used in the production oflight. After incubation for 20 h, Apoptotic activity was assessed bymeasure levels of aminoluciferin. % Apoptosis was calculated from foldchange in luminescence compared to DMSO treated sample.

Example 9. FIG. 11 Shows that the Cmd 1 Causes Selective and EnhancedInduction of ISG and PRR-Associated Genes in Acute Monocytic LeukemiaCell Line (THP1) Compared to Primary Cells PBMCs. Gene ExpressionAnalysis in THP1 and PBMCs

THP1 cells and PBMCs grown in complete media were treated with 5 uM ofeither Cmd 1 or 2′3-cGAMP or DMSO control with Lipofectamine LTX. Afterincubation for 20 h, RNA was extracted and gene expression of differentInterferon Stimulated Genes (ISGs) and various Pattern RecognitionReceptors (PRRs) was evaluated by real time PCR. Fold Induction wascalculated by ΔΔct method.

Example 10. FIG. 12 Shows that Cmd 1 Inhibits Tumor Cell Growth

Tumor cells in 96-well plate were treated once daily with Cmd 1 (nolipofectamine) or recombinant IFN (U-IFN) for 3 days. Cells were fixedwith 1% paraformaldehyde and stained with DAPI. Cells were automaticallyimaged on ImageXpress and total number of survival cells were analyzedusing MetaXpress software. Results are shown as total number of cellsper group or % reduction calculated by normalizing to DMSO treatedcells.

Example 11. FIG. 21 Shows that Cmd 4 has Enhanced Activity in AcuteMonocytic Leukemia Cell Line (THP1) Compared to Primary Cells PBMCs

Gene expression analysis in THP1 and PBMCs: THP1 cells and PBMCs grownin complete media were treated with 5 uM of either Cmd 4 or 2′,3′-cGAMPor DMSO control with Lipofectamine LTX. After incubation for 20 h, RNAwas extracted and gene expression of different Interferon StimulatedGenes (ISGs) and various Pattern Recognition Receptors (PRRs) wasevaluated by real time PCR. Fold Induction was calculated by ΔΔctmethod.

Example 12. Efficacy of Exemplary Compounds Via IntraperitonealAdministration in a Breast Carcinoma Model

The efficacy of intraperitoneal administration of Cmd 1 was investigatedin the 4T1.luc2 orthotopic murine breast carcinoma model. Thirty femaleBALB/c mice between 7-10 weeks old were randomized into four treatmentgroups based on Day 1 body weight, and the treatment was carried outaccording to the regimen outlined in Table 4 below. Cmd 1 was dissolvedin saline and administered at 10 mL/kg (0.200 mL/20 g mouse), with acell injection volume of 0.05 mL/mouse.

TABLE 4 IP administration in breast carcinoma model: study regimenRegimen 1 Gr. N Agent mg/kg Route Schedule 1 10 Vehicle — ip days 5, 7,9, 11, 14, 18 2 10 Cmd 1 10 ip days 5, 7, 9, 11, 14, 18 3 5 Vehicle — ipdays 5, 7, 9, 11, 14, 18 4 5 Cmd 1 10 ip days 5, 7, 9, 11, 14, 18

Each animal was monitored individually. The endpoint of the experimentwas a tumor volume of 2000 mm2 or 45 days. Animals in Groups 1 and 2were subjected to whole body bioluminescent imaging starting on Day 5and once a week thereafter (Days 12, 19, 26, 33, and 41). At theendpoint, blood and tissue (lung, lymph nodes, spleen, and tumor) wasanalyzed for presence of metastases and biomarker (CD45, CD3, CD4, CD8,CD11b, CD25, Ly-6G, Ly-6C, FoxP3) levels. As seen in FIG. 70 , micetreated with Cmd 1 showed a significant decrease in tumor growthcompared with control

Example 13. Determination of Maximum Tolerated Dose of OrallyAdministered Exemplary Compounds

In order to investigate the maximum tolerated dosage of orallyadministered compounds, 15 female BALB/c mice between 7-10 weeks oldwere split into three treatment groups. Each group was administeredeither Cmd 1 or vehicle orally, according to the schedule outlined inTable 5 below. Cmd 1 was provided at 10 mL/kg (0.200 mL/20 g mouse).Upon oral administration of Cmd 1, once daily or twice daily up to 60mgkg/day, there were no adverse clinical signs and the compound was welltolerated as shown in Table 5.

TABLE 5 Oral MTD study regimen and results Treatment Regimen 1 TreatmentRegimen 2 BW Mean Da Group n Agent Vehicle mg/kg Route Schedule VehicleRoute Schedule Nadir TR NTR NTRm of Death 1 5 Cmd 1 60 po qd × 10 — — —— 0 0 0 — — 2 5 Cmd 1 60 po bid × 10 — — — −3.6% 0 0 0 — first day (11)1 dose 3 5 saline — po qd × 9  ip qd × 1 — 0 1 0 — (start on — day 10)

EQUIVALENTS

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated by reference in theirentirety. While this disclosure has been described with reference tospecific aspects, it is apparent that other aspects and variations maybe devised by others skilled in the art without departing from the truespirit and scope of the disclosure. The appended claims are intended tobe construed to include all such aspects and equivalent variations. Anypatent, publication, or other disclosure material, in whole or in part,that is said to be incorporated by reference herein is incorporatedherein only to the extent that the incorporated material does notconflict with existing definitions, statements, or other disclosurematerial set forth in this disclosure. As such, and to the extentnecessary, the disclosure as explicitly set forth herein supersedes anyconflicting material incorporated herein by reference.

While this disclosure has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the disclosureencompassed by the appended claims.

What is claimed is:
 1. A compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 2. The compound of claim1, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 3. The compound of claim1, wherein the compound is


4. The compound of claim 1, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 5. The compound of claim1, wherein the compound is


6. The compound of claim 1, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 7. The compound of claim1, wherein the compound is


8. A method of treating cancer, comprising administering to a subject inneed thereof an effective amount of a compound of claim 1 or apharmaceutically acceptable salt thereof, wherein the cancer is acutemonocytic leukemia, lymphoma, melanoma, colon cancer, or breast cancer.9. The method of claim 8, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 10. The method of claim8, wherein the compound is


11. The method of claim 8, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 12. The method of claim8, wherein the compound is


13. The method of claim 8, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 14. The method of claim8, wherein the compound is


15. The method of claim 8, wherein the cancer is colon cancer.
 16. Themethod of claim 8, wherein the cancer is breast cancer.
 17. The methodof claim 16, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 18. The method of claim16, wherein the compound is


19. The method of claim 16, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 20. The method of claim16, wherein the compound is


21. The method of claim 16, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 22. The method of claim16, wherein the compound is